Systems and methods for fractionation and collection of analytes in a sample

ABSTRACT

Embodiments include systems, apparatuses, and methods to efficiently separate analytes in a sample and elute fractions of the separated analytes. In some embodiments, a method includes introducing a sample in a capillary with a first end ionically coupled to a first running buffer and a second end ionically coupled to a second running buffer to form a pH gradient. The method includes applying a voltage between the first running buffer and the second running buffer, to separate a plurality of analytes in the sample. The method includes disposing the second end of the capillary in a collection well including a chemical mobilizer and applying a voltage to elute one or more analytes from the plurality of analytes in the sample, that have been separated, into the collection well. Embodiments include detection methods to monitor separation of analytes, mobilization of analytes, and/or elution of fractions containing analytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a divisional of U.S. patentapplication Ser. No. 17/463,326, filed on Aug. 31, 2021, the content ofwhich is incorporated by reference herein in its entirety.

BACKGROUND

The embodiments described herein relate generally to systems and methodsfor separating, analyzing, and eluting fractions from sample mixtures.Some embodiments described herein relate to separation and/orfractionation of an analyte or analytes present in a sample based on aproperty. More particularly, the embodiments described herein generallyrelate to systems and methods configured to perform capillary-basedfractionation using electrophoretic methods of separation followed byelution.

SUMMARY

Systems and methods for capillary electrophoresis, analyte visualizationand fraction collection are described herein. In one aspect, a methodincludes introducing, at a first time, a sample containing a pluralityof analytes in a conductive medium into a capillary. The method includesionically coupling a first end of the capillary to a first runningbuffer having a first pH, and ionically coupling a second end of thecapillary to a second running buffer having a second pH, such that a pHgradient forms along the capillary. The method further includesseparating, at a second time after the first time, at least a subset ofthe plurality analytes according to their isoelectric points by applyinga voltage across the first running buffer and the second running bufferwhen the first end of the capillary is ionically coupled to the firstrunning buffer and the second end of the capillary is ionically coupledto the second running buffer. The method further includes detecting ananalyte from the plurality of analytes separated along the pH gradient.The method further includes identifying a peak of a distributionassociated with an amount of separated analyte along the pH gradient.The method further includes placing the second end of the capillary intoa well including a chemical mobilizer at a third time after the firsttime to mobilize and selectively elute an analyte from the plurality ofanalytes from the capillary and into the well, based on the identifyingthe peak of the distribution associated with an amount of separatedanalyte. The method optionally includes monitoring a migration of thepeak of distribution associated with the amount of separated analyteduring elution to collect an individual fraction of the separatedanalyte

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a fractionation system, a systemconfigured to perform capillary electrophoresis and/or fractionateanalytes separated by capillary electrophoresis, according to anembodiment.

FIG. 2 is a schematic of a side view of an example capillary cartridgethat can be used with a fractionation system, according to anembodiment.

FIGS. 3 is a schematic of a side view of an example capillary cartridgethat can be used with a fractionation system, according to anembodiment.

FIG. 4 is an image of a side view of a capillary cartridge that can beused with a fractionation system, according to an embodiment.

FIG. 5 is a schematic of a side view of a portion of an examplecapillary cartridge that can be used with a fractionation system,according to an embodiment.

FIG. 6 is a perspective view of a fractionation system, according to anembodiment.

FIGS. 7A and 7B are perspective views of sample plate assembliesconfigured to be used with a fractionation system, according to twoexample embodiments.

FIG.8 is a flowchart of a method of using a fractionation system,according to one implementation.

FIGS. 9A, 9B, and 9C are plots of experimental data indicatingseparation of an analyte using a standard separation system, afractionation system with a single segment capillary, and afractionation system using a multi-segment capillary, respectively.

FIG. 10 depicts experimental data indicating a mobilization of ananalyte using a fractionation system.

FIG. 11 depicts experimental data indicating a mobilization of ananalyte using a fractionation system.

FIG. 12 depicts experimental data indicating a separation andmobilization of analytes using a fractionation system.

FIG. 13 depicts experimental data indicating fractions of a samplemixture collected using a fractionation system.

FIG. 14 depicts experimental data indicating fractions of a samplemixture collected using a fractionation system.

FIG. 15 depicts experimental data indicating fractions of a samplemixture collected using a fractionation system.

FIG. 16 depicts experimental data of mass spectra obtained fromfractions of a sample mixture collected using a fractionation system.

FIG. 17 depicts experimental data indicating fractions of a samplemixture collected using a fractionation system.

FIG. 18 depicts experimental data indicating fractions of a samplemixture collected using a fractionation system.

DETAILED DESCRIPTION

In some embodiments, a method includes introducing, at a first time, asample containing a plurality of analytes in a conductive medium into acapillary. The method includes ionically coupling a first end of thecapillary to a first running buffer having a first pH, and ionicallycoupling a second end of the capillary to a second running buffer havinga second pH, such that a pH gradient forms along the capillary. Themethod further includes separating, at a second time after the firsttime, at least a subset of the plurality analytes according to theirisoelectric points by applying a voltage across the first running bufferand the second running buffer when the first end of the capillary isionically coupled to the first running buffer and the second end of thecapillary is ionically coupled to the second running buffer. The methodfurther includes placing the second end of the capillary into a wellincluding a chemical mobilizer at a third time after the first time toselectively elute an analyte from the plurality of analytes from thecapillary and into the well.

In some embodiments, a method includes introducing, at a first time, asample in a conductive medium into a capillary, a first end of thecapillary being ionically coupled to a first running buffer having afirst pH, a second end of the capillary being ionically coupled to asecond running buffer having a second pH such that a pH gradient isformed along the capillary. The method further includes separating, at asecond time after the first time, a plurality of analytes from thesample by applying a voltage between the first running buffer and thesecond running buffer. The method further includes moving, at a thirdtime after the second time, the second end of the capillary from areservoir containing the second running buffer to a collection well, andincreasing, at a fourth time after the third time, a pressure at thefirst end of the capillary to elute a portion of the sample containing afirst analyte from the plurality of analytes that has a firstisoelectric point and not a second analyte from the plurality ofanalytes that has a second isoelectric point different from the firstisoelectric point.

In some embodiments, an apparatus includes a capillary configured toelectrophoretically focus an analyte contained within a sample, arunning buffer reservoir configured to contain a first running bufferhaving a first pH in ionic communication with a first end of thecapillary, and a sample plate defining a plurality of wells. The sampleplate and the capillary are collectively configured such that a secondend of the capillary can move between each well from the plurality ofwells. The apparatus includes a first well from the plurality of wellsthat is configured to contain the sample, and the capillary and thesample plate are collectively configured such that, when a second end ofthe capillary is disposed in the first well, a portion of the sample canbe drawn into the capillary. The apparatus includes a second well fromthe plurality of wells that is configured to contain a second runningbuffer having a second pH different from the first pH such that when thesecond end of the capillary is disposed in the second well, the bufferreservoir containing the first running buffer and the second wellcontaining the second running buffer are in ionic communication. Theapparatus further includes an electrical power source configured toapply a voltage across the running buffer reservoir and the secondrunning buffer such that, when the second end of the capillary isdisposed in the second well and the voltage is applied, a pH gradient isestablished across the capillary and the analyte migrates to a portionof the capillary associated with its isoelectric point. The apparatusincludes a third well from the plurality of wells that is configured tocontain a chemical mobilizer.

A chemical mobilizer can be a buffered solution configured to supplyions to the capillary, typically after a steady state pH gradient hasbeen established and analytes have been focused into stationarypositions in the capillary and the pH gradient corresponding to theirisoelectric points. The chemical mobilizer disrupts the (typicallysteady state) pH gradient and in doing so imparts a charge on theanalyte causing it to migrate. Such analytes can migrate into collectionwells containing the chemical mobilizer. Chemical mobilizers generallyare operable to cause the separated analytes to migrate and/or beeluted. In some implementations, multiple collection wells containing achemical mobilizer can be used to successively move the distal end ofthe capillary to be disposed in each successive collection well suchthat separated analytes can be eluted in the order in which they arefocused. In some implementations, a chemical mobilizer can be used tocompletely elute all separated analytes into a set of collection wellsor vials. In some implementations, a chemical mobilizer can be used topartially elute a portion of the separated analytes into a set ofcollection wells or vials.

Techniques such as Isoelectric focusing (IEF) can be a powerful approachto separating analytes in a sample, for example, charge variants ofprotein molecules such as monoclonal antibody (mAb) or other biologicalmolecules, with good resolution and sensitivity. Therapeutic monoclonalantibodies (mAbs) make up a large portion of the rapidly growing drugmarket. Ensuring safety and efficacy through comprehensive understandingof these products' critical quality attributes (CQAs), including chargeheterogeneity, is a regulatory requirement. Various charge isoforms ofmAbs can result from cell culture or production processes, potentiallyaffecting the mAb structure and function. Imaged capillary isoelectricfocusing (icIEF) is a method that can be used for charge profiling.Ion-exchange chromatography (IEC) has also been a major tool forfractionation combined with characterization. IEC, however, is notcompatible with certain types of molecules, hydrophobic antibody drugconjugates (ADCs) for example, and icIEF typically provides higherseparation resolution. Moreover, an individual charge variant obtainedfrom IEC fractionation may not be comparable to the variant peak in theicIEF profile. Therefore, there is an unmet need for IEF-basedfractionation of charge variants for characterization.

IEF can be performed with the sample mixed with ampholytes sandwichedbetween an acid and a base reservoir. Under an electric field, eachcharged component of the sample migrates to a position along a pHgradient formed by the ampholytes where the pH is the same as thatcomponent's isoelectric point (pI). Capillary isoelectric focusing(CIEF) is a variant of this approach where IEF is performed in a sampleheld in a lumen of a capillary.

In case of CIEF, due to the miniatured fluidic path and insignificantJoule heating involved, larger magnitude electric fields can be appliedfor the separation of components in a sample held in the lumen of acapillary, resulting in fast separation and better resolution ofseparation of the analytes in the sample. Whole column imaging capillaryisoelectric focusing (iCIEF) is a method that can used as described inU.S. Patent Application Publication No. 10,794,860 entitled, “Systemsand methods for capillary electrophoresis, isoelectric point, andmolecular weight analysis,” filed on Jul. 12, 2018, the disclosure ofwhich is incorporated herein by reference in its entirety. iCIEF furtherimproves the speed, resolution, and precision of the assay due to thefact that no sample mobilization is needed for the detection and ashorter capillary can be used for the separation. Because of itssuperior performance, iCIEF can be widely used as a standardizedanalytic tool in several industries including pharmaceutical industryfor characterization and quality control of therapeutic proteinsincluding mAb, antibody drug conjugate (ADC) and other biologicalmolecules.

While iCIEF is a powerful method yielding rich information about chargevariants of proteins, sometimes additional information may be desired tofully characterize and identify a molecule, for example, to identifyand/or isolate unknown impurities that may arise from formulation orbioprocessing stages of generating the molecule. It may be desirable tonot only separate the charge variants but also to isolate them forfurther analysis using the methods such as Mass Spectrometry (MS) orother biological assays.

Methods explored to utilize additional processing in conjunction withseparation techniques like iCIEF can be categorized into two groups:fraction collection and hyphenated cIEF-MS. Fraction collection methodsallow individual fractions of charge variants in a sample to becollected and further processed according to desired needs. HyphenatedcIEF-MS methods interface directly from the capillary of the cIEF systeminto the ionization source of a MS system. While the hyphenated cIEF-MSmethod circumvents the efforts needed for fraction collection, it hassome limitations: (1) the fractions cannot be analyzed by downstreamanalysis method other than MS; (2) performance of the MS will becompromised if the cIEF run needs UREA or any other additives that areunfriendly to MS systems; (3) peptide mapping is not possible on suchhyphenated platform. On the other hand, multiple fraction collectiondevices have been developed and commercialized but with limited successeither because of the poor performance (e.g., poor resolution,insufficient sensitivity, low yield, etc.) or because they are difficultto operate (e.g., complicated device set up, lack of robustness, etc.).Embodiments disclosed herein provide a novel icIEF fractionationsolution, which involves icIEF separation and collection of chargevariants. Some embodiments described enable MauriceTM icIEF-based peakidentification followed by downstream characterization, such as nativeanalysis of collected charge variants using ZipChip (CE-ESI) due to thebroad sample matrix compatibility, easy sample prep, and fast massspectrometry analysis time.

Embodiments described herein include apparatus, methods, and systems forperforming fractionation of analytes in a sample using a suitableseparation technique (e.g., capillary isoelectric focusing) such thatthere is streamlined, semi-automatic, separation, visualization,detection, and/or fractionation of analytes in a sample into fractioncollection wells. The fractionation can be such that the collectedanalytes can be further processed using any suitable technique withoutany restriction as in the case of the hyphenated cIEF-MS methods.

Definitions

As used in this specification, the singular forms “a,” “an” and “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, the term “a member” is intended to mean a singlemember or a combination of members, “a material” is intended to mean oneor more materials, or a combination thereof.

As used herein, the terms “about” and “approximately” mean plus or minus10% of the value stated and all values in between. For example, about0.5 would include 0.45 and 0.55, about 10 would include 9 to 11, about1000 would include 900 to 1100. The term “substantially” when used inconnection with shape relationships (e.g., linear, cylindrical, etc.),structural relationships (e.g., perpendicular, etc.), and/or othergeometric relationships is intended to convey that the structure sodefined is nominally linear, cylindrical, perpendicular, and/or thelike. As one example, a portion of a support member that is described asbeing “substantially linear” is intended to convey that, althoughlinearity of the portion is desirable, some non-linearity can occur in a“substantially linear” portion. Such non-linearity can result frommanufacturing tolerances, or other practical considerations (such as,for example, the pressure or force applied to the support member). Thus,a geometric construction modified by the term “substantially” includessuch geometric properties within a tolerance of plus or minus 5% of thestated geometric construction. For example, a “substantially linear”portion is a portion that defines an axis or center line that is withinplus or minus 5% of being linear.

As used herein, the terms “perpendicular” and/or “normal” generallydescribe a relationship between two geometric constructions (e.g., twolines, two planes, a line and a plane, or the like) in which the twogeometric constructions are disposed at substantially 90°. For example,a line is said to be “perpendicular” to another line when the linesintersect at an angle substantially equal to 90°. Similarly, when aplanar surface (e.g., a two-dimensional surface) is said to be “normal”to another planar surface, the planar surfaces are disposed atsubstantially 90° (e.g., substantially orthogonal) as the planarsurfaces extend to infinity.

As used herein the term “module” refers to any assembly and/or set ofoperatively-coupled electrical components that can include, for example,a memory, a processor, electrical traces, optical connectors, software(executed in hardware), and/or the like. For example, a module executedin the processor can be any combination of hardware-based module (e.g.,a field-programmable gate array (FPGA), an application specificintegrated circuit (ASIC), a digital signal processor (DSP)) and/orsoftware-based module (e.g., a module of computer code stored in memoryand/or executed at the processor) capable of performing one or morespecific functions associated with that module.

As used herein, the terms “analyte” and/or “target analyte” refer to anymolecule or compound to be separated and/or detected with the methods,apparatus and systems provided herein. Suitable analytes include, butare not limited to, small chemical molecules such as, for example,environmental molecules, clinical molecules, chemicals, pollutants,and/or biomolecules. More specifically, such chemical molecules caninclude, but are not limited to pesticides, insecticides, toxins,therapeutic and/or abused drugs, antibiotics, organic materials,hormones, antibodies, antibody fragments, antibody-molecule conjugates(e.g., antibody-drug conjugates), antigens, cellular membrane antigen,proteins (e.g., enzymes, immunoglobulins, and/or glycoproteins), nucleicacids (e.g., DNA and/or RNA), lipids, lectins, carbohydrates, wholecells (e.g., prokaryotic cells such as pathogenic bacteria and/oreukaryotic cells such as mammalian tumor cells), viruses, spores,polysaccharides, glycoproteins, metabolites, cofactors, nucleotides,polynucleotides (comprising ribonucleic acid and/or deoxyribonucleicacid), transition state analogs, inhibitors, receptors, receptor ligands(e.g., neural receptors or their ligands, hormonal receptors or theirligands, nutrient receptors or their ligands, and/or cell surfacereceptors or their ligands), receptor-ligand complexes, nutrients,electrolytes, growth factors and other biomolecules and/ornon-biomolecules, as well as fragments and combinations thereof. In someembodiments, the analyte is a protein or a protein complex, and thesample is a cellular lysate or a purified protein. Other suitableanalytes can include aggregates, agglomerates, floc, and/or dispersedphase droplets or particles of colloids and/or emulsions.

As used herein, the term “sample” refers to a composition that containsan analyte or analytes to be detected. A sample, in some embodiments, isheterogeneous, containing a variety of components (e.g., differentproteins) or homogenous, containing one component (e.g., a population ofone protein). In some instances, a sample can be naturally occurring, abiological material, and/or a manufactured material. Furthermore, asample can be in a native (e.g., a cell suspension) or denatured form(e.g., a lysate). In some instances, a sample can be a single cell (orcontents of a single cell, e.g., as a cellular lysate from the singlecell, or a purified protein) or multiple cells (or contents of multiplecells, e.g., as a cellular lysate from the multiple cells, or a purifiedprotein from the multiple cells), a blood sample, a tissue sample, askin sample, a urine sample, a water sample, and/or a soil sample. Insome instances, a sample can be from a living organism, such as aeukaryote, prokaryote, mammal, human, yeast, and/or bacterium or thesample can be from a virus.

In some embodiments, the sample is a heterogeneous biological sample orderived from a heterogeneous biological sample, for example a tissuelysate, a cellular lysate or a mixture of biomolecules such as proteins(e.g., a purified protein). In a further embodiment, a protein withinthe cellular lysate is the analyte to be detected by the methods andsystems described herein. In a further embodiment, the apparatus,systems, and methods provided herein provide for the detection of aparticular form of a protein, for example, a phosphorylated protein. Thecellular lysate, for example, can be the lysate of one cell or a mixtureof cells. Moreover, the cellular lysate can include a single cell type,or multiple cell types. The cell type, in some embodiments, includes astem cell or a cancer cell, or a population of stem cells, or apopulation of cancer cells. In some embodiments, a sample comprises oneor more stem cells (e.g., any cell that has the ability to divide forindefinite time periods and to give rise to specialized cells) and/orstem cell lysates. Suitable examples of stem cells can include but arenot limited to embryonic stem cells (e.g., human embryonic stem cells(hES)), and non- embryonic stems cells (e.g., mesenchymal,hematopoietic, induced pluripotent stem cells (iPS cells), or adult stemcells (MSC)).

In some instances, prior to detecting and/or fractionating an analyte ina sample with the apparatus and systems provided herein, processing maybe performed on the sample. For example, a sample can be subjected to alysing step, denaturation step, heating step, purification step (e.g.,protein purification), precipitation step, immunoprecipitation step,column chromatography step, centrifugation, etc. In some embodiments, asample is subjected to a denaturation step prior detecting and/orseparating a target analyte in a sample with the methods, apparatus, andsystems described herein. The processing step on the sample, in someembodiments, is performed in one of the apparatus or systems describedherein. In another embodiment, the processing step is performed prior tointroducing the sample into one of the apparatus or systems set forthherein.

As used herein, the terms “standard” and/or “internal standard” refer toa well-characterized substance of known amount and/or identity (e.g.,known isoelectric point, molecular weight, electrophoretic mobilityprofile, number of base pairs in the case of a nucleic acid, molecularcomposition, etc.) that can be added to a sample comprising the analyte,for comparative purposes. In some embodiments, a known quantity ofstandard is added to a sample comprising one or more analytes, and boththe standard and the molecules in the sample, including the analyte(s)are separated on the basis of isoelectric point by electrophoresis). Acomparison of the standard and analyte signal then provides aquantitative or semi-quantitative measure of the amount of analyteoriginally present in the sample.

In general, isoelectric focusing (IEF) standards are known based onestablished isoelectric point. Similarly, molecular weight standards areknown. In some instances, the standard and/or the analyte(s) can bedetected with one or more detection molecules or reagents, such as withan antibody against the analyte or a labeling moiety attached to thestandard. In some embodiments, a primary antibody is used to bind thetarget analyte, and a secondary antibody conjugated to a fluorescent ora chemiluminescent reagent is introduced to bind the primary antibody orthe primary antibody-analyte complex. The signal of the fluorescent orchemiluminescent molecule is then detected. In other instances, thestandard and/or the analyte(s) can be detected via native fluorescence(e.g., via fluorescence of tryptophan amino acids within the standardand/or analyte(s)) and/or absorbance. The signal of the standard and thesignal of the analyte(s) can then be compared to measure theconcentration of the analyte(s) in the sample. In addition, oralternatively, a relevant characteristic of the analyte (e.g.,isoelectric point, molecular weight, etc.) can be determined bycomparison to the standard.

In some embodiments, an internal standard can be a purified form of theanalyte itself, which is generally made distinguishable from the analytein some way. Any method of obtaining a purified form of the analyte caninclude but is not limited to purification from nature, purificationfrom organisms grown in the laboratory (e.g., via chemical synthesis),and/or the like. The distinguishing characteristic of an internalstandard can be any suitable change that can include but is not limitedto dye labeling, radiolabeling, or modifying the mobility of thestandard during the electrophoretic separation so that it isdistinguishable from the analyte. For example, the analyte and theinternal standard can each be labeled with fluorescent dyes that areeach detectable at discrete emission wavelengths, thereby allowing theanalyte and the standard to be independently detectable. In someinstances, an internal standard is different from the analyte butbehaves in a way similar to or the same as the analyte, enablingrelevant comparative measurements. In some embodiments, a standard thatis suitable for use can be any of those described in U.S. PatentApplication Publication No. 2007/0062813 entitled, “ElectrophoresisStandards, Methods and Kits,” filed on Sep. 20, 2006, the disclosure ofwhich is incorporated herein by reference in its entirety. For example,in some embodiments, the multiple analytes are a population of proteinsor a subpopulation of proteins. In this regard, it may not be practicalto include a single internal standard corresponding to each of theindividual proteins of the population of proteins or subpopulation ofproteins. Accordingly, in some embodiments, a general isoelectric pointstandard is introduced into the systems and apparatus provided herein.The standard, in some embodiments, can be a ladder standard operable toidentify different isoelectric points along the capillary tube. Proteinsin the sample that migrate during the electrophoresis are compared tothe ladder to determine the isoelectric point of the proteins present inthe sample. In some embodiments, ladder standards are used.

Overview of a Fractionation Process

Embodiments described include systems and methods to perform separation,detection, and/or fractionation of one or more analytes in a sample(e.g., based on molecular weight and/or isoelectric point). The samplecan be prepared in a conductive medium and loaded into capillary that isin turn loaded into a fractionation system (also referred to herein as“the system”).

The embodiments of fractionation systems described herein can be used toseparate analytes, detect and/or visualize separated analytes, andselectively fractionate one or more analytes in a sample, based on theseparation and/or visualization, using a single system. Embodimentsdescribed herein can use microfluidic separation techniques, therebyenabling the analysis of very small volume samples.

In some instances, multiple analytes can be separated, detected, and/orfractionated from a sample loaded in a single capillary by the systemusing apparatus and/or methods provided herein. For example, in someinstances, a user can load a capillary cartridge into the system and caninitiate and/or otherwise provide instructions to the system to causethe system to at least semi-automatically separate analytes (e.g.,proteins) within the sample by isoelectric point.

Separation

Analytes and/or standards described above, can be separated using afractionation system by taking advantage of any suitable mobilityparameter such as charge, molecular weight, electrophoretic mobility(e.g., influenced by molecular weight, characteristic length, area, orvolume, oligonucleotide length, or other suitable characteristic),and/or the like. The sample can be loaded into a capillary and thecapillary can be positioned in the system such that a first end of thecapillary is coupled to a first running buffer having a first pH and asecond end of the capillary can be ionically coupled to a second runningbuffer having a second pH, such that a pH gradient is formed along thelength of the capillary via the lumen of the capillary. The analytes canbe separated (e.g., according to their isoelectric points) by applying avoltage across the first running buffer and the second running bufferFor example, in some embodiments a voltage can be applied between thefirst running buffer having the first pH that is ionically coupled to afirst end of the capillary, and a second running buffer having thesecond pH that is ionically coupled to a second end of the capillary.The applying of the voltage across the ends of the capillary can induceseparation of analytes along a fluid path in the capillary lumencomprising the sample, based on a mobility parameter such as anisoelectric point and/or the like.

In some embodiments, the capillary can include a separation matrix,which can be added in an automated fashion. Capillary electrophoresis ina separation matrix using the system can be analogous to separation in apolymeric gel, such as a polyacrylamide gel or an agarose gel, wheremolecules are separated on the basis of the mobility parameter of themolecules in the sample, by providing a porous passageway of fluid paththrough which the molecules can travel.

Detection

In some embodiments, once the separation is complete, and the separatedanalytes can be probed and analyzed to determine an identity of ananalyte and/or to determine a degree of separation of analytes. In otherwords, once the analytes and/or standards are separated, the apparatusand/or systems described herein can continue to provide a flow ofelectric current and/or a pressure differential (e.g., using a vacuumsource) operable to maintain the analytes and/or standards at theirrespective relative points of separation (e.g., isoelectric points). Insome instances, a pressure differential (e.g., negative pressure) can beapplied to offset gravitational effects due to a vertical column ofliquid which may otherwise create a hydrodynamic flow that may reduceresolution and/or mobilize the pH gradient formed in the capillary. Thefractionation system can be initiated to or instructed to probe andanalyze the contents of one or more portions of the capillary lumencomprising the separated analytes based on a property (e.g., isoelectricpoint). The system can probe using suitable detectors via viewingwindows that provide access to the capillary lumen. In some instances,the system captures digital or analog images associated with a detectionand/or separation of analytes within a sample (e.g., including anysuitable agent, reagent, protein, analyte, buffer, lysate, etc.) drawninto a capillary of the capillary cartridge. The system can analyze theimages and/or other data associated with the detection and can provide ameasure of relative separation of the constituents of the sample. Thesystem can use the measure of relative separation between the analytesand/or constituents to determine a desired rate of migration of theseparated constituents of the sample towards a distal end and to eluteone or more fractions of the separated analytes.

In some embodiments, the sample can include or be subjected to detectionagents that may be bound to the separated analytes and/or standards suchthat they can be then probed to identify and/or localize each separatedanalyte and/or standard. A detection agent can be capable of binding toor interacting with the analyte and/or standard to be detected.Detection agents may allow the detection of a standard and an analyte byany means such as but not limited to fluorescent dye(s), optical dye(s),chemiluminescent reagent(s), radioactivity, particles, magneticparticle(s), paramagnetic particle(s), etc. Detection agents can includeany organic or inorganic molecules such as, for example, proteins,peptides, antibodies, enzyme substrates, transition state analogs,cofactors, nucleotides, polynucleotides, aptamers, lectins, smallmolecules, ligands, inhibitors, drugs, and other biomolecules as well asnon-biomolecules capable of binding the analyte to be detected. In someembodiments, the detection agents comprise one or more label moieties.Label moieties can be a reactive moiety that includes a functional groupthat can be converted to a functionality that adheres to an analyte viaany suitable interaction including hydrophobic interactions, ionicinteractions, hydrogen bonding and/or the like. In some embodiments,such reactive moieties can be activated by light (e.g., UV light), lasermediated excitation, temperature mediated excitation, or any othersource of energy in order to label one or more analytes separated and/orlocalized in the fluid paths in a capillary. In some embodiments, thedetection agents can include one or more label moiety(ies). Inembodiments employing two or more label moieties, each label moiety canbe the same, or some, or all, of the label moieties may differ.

In some embodiments, the label moiety can be and/or can include achemiluminescent label. Suitable labels moieties can include enzymescapable of reacting with a chemiluminescent substrate in such a way thatphoton emission by chemiluminescence is induced. For example, enzymescan induce chemiluminescence in other molecules through enzymaticactivity. Such enzymes can be and/or can include peroxidase, forexample, horseradish peroxidase (HRP), β-galactosidase, phosphatase,etc. In some embodiments, the chemiluminescent label can be selectedfrom any of a variety of classes of luminol label, an isoluminol label,etc. In some embodiments, a detection agent can includechemiluminescent- labeled antibodies, for example, a secondary antibodycovalently bound to HRP. In some embodiments, the detection agentscomprise chemiluminescent substrates such as, for example, Galactonsubstrate available from Applied Biosystems of Foster City, Californiaor SuperSignal West Femto Maximum Sensitivity substrate available fromPierce Biotechnology, Inc. of Rockford, Illinois, or any other suitablesubstrates. In some embodiments, a detection agent can be any of thosedescribed in U.S. Pat. Nos. 6,689,576, 6,395,503, 6,087,188, 6,287,767,6,165,800, and 6,126,870, the disclosures of which are incorporatedherein by reference in their entireties.

In some embodiments, the label moiety can be and/or can include abioluminescent compound (e.g., found in biological systems in which acatalytic protein increases the efficiency of the chemiluminescentreaction). The presence of a bioluminescent compound is determined bydetecting the presence of luminescence. Suitable bioluminescentcompounds include, but are not limited to luciferin, luciferase, andaequorin.

In some embodiments, the label moiety can be and/or can include afluorescent dye. Such fluorescent dyes can include aresonance-delocalized system or aromatic ring system that absorbs lightat a first wavelength and emits fluorescent light at a second wavelengthin response to the absorption event. Fluorescent dyes can be any of avariety of classes of fluorescent compounds such as but not limited toxanthenes, rhodamines, fluoresceins, cyanines, phthalocyanines,squaraines, bodipy dyes, coumarins, oxazines, and carbopyronines. Insome embodiments, for example, where detection agents containfluorophores, such as fluorescent dyes, their fluorescence is detectedby exciting them with an appropriate light source, and monitoring theirfluorescence by a detector sensitive to their characteristicfluorescence emission wavelength.

In some embodiments, the label moiety can be and/or can include aphotopigment with a specified optical density that can used todistinguish regions of localization of the analyte bound to thephotopigment (e.g., based on one or more peaks in optical density signalfrom probing the fluid path in a capillary) from regions of separationbetween analytes (e.g., based on a trough in the optical density signalfrom detection).

In some embodiments, two or more different agents can be used to bind toor interact with two or more different analytes to enable more than onetype of analytes to be detected simultaneously. In some embodiments, twoor more different detection agents, which bind to or interact with theone analyte, can be detected simultaneously. In various embodiments,using two or more different detection agents, one agent can bind to orinteract with one or more analytes to form a first agent-analytecomplex, and a second reagent, the detection agent can be used to bindto or interact with the first agent-analyte complex.

In another embodiment, two or more different detection agents can enabledetection of two or more forms of analytes of interest. In someembodiments, a single specific detection agent, for example an antibody,can allow detection and analysis of both phosphorylated andnon-phosphorylated forms of an analyte. In some embodiments, multipledetection agents can be used with multiple substrates to provide colormultiplexing. For example, different chemiluminescent substrates can beused to emit light of differing wavelengths. Selective detection ofdifferent emission wavelengths and/or emitted colors (e.g., via adiffraction grating, a prism(s), a series of colored filters, and/or thelike) can allow determination of which color photons or light of whatrange of wavelengths is being emitted at any position along a fluid path(e.g., along a molecular weight gradient), and therefore determinationof which detection agents are present at each emitting location in thecapillary lumen. In some embodiments, selective detection of differentemission wavelengths associated with different analytes can be used todetermine the emitting location of an analyte and follow a path and/orrate of migration of the analyte as the separated analytes and/orstandards are guided to migrate towards a distal end of the capillary tobe eluted and/or fractionated. In some embodiments, differentchemiluminescent reagents can be supplied sequentially, allowingdifferent bound detection agents to be detected sequentially.

Visualization and Elution

In some embodiments, the separated and/or labeled analytes can bevisualized and their relative localization along the fluid path can bedetermined. The apparatus can then be manipulated to elute one or moreanalytes individually based on the visualization and/or relativelocalization of the separated analytes. For example, one or more of theseparated analytes can be mobilized towards a distal end of the fluidpath and eluted out of the fluid path with careful isolation using anysuitable driving force (e.g., pressure ejection, delivering varyingvoltages between a proximal end of the capillary and the distal end ofthe capillary, chemical mobilizers, etc.). That is, followingvisualization one or more of the separated analytes can be made tomigrate towards a distal end of the capillary, and eluted out of thecapillary to be collected in a collection well. The separated analytescan be mobilized towards the distal end of the capillary using anysuitable technique including pressure ejection, voltage inducedmobilization towards a distal end of the capillary, elution usingchemical mobilizers, etc. In some instances, chemicals with differentnegative ions, for example, acetate and phosphate, can be used aschemical mobilizers.

In some embodiments, the analytes and/or standards that have beenseparated can be mobilized and/or caused to migrate at a desired rate. Arate of migration and/or a rate mobilization of the separated analytescan be manipulated using any suitable mechanism including appliedelectric current, applied pressure, etc. In some implementations, theseparation of the analytes can be continuously monitored duringmobilization and/or elution. Mobilization and/or elution can beaccomplished while maintaining at least a minimum or desired degree ofrelative separation based on their respective isoelectric points (e.g.,a minimal separation between peaks or distributions of two adjacentseparated analytes may be maintained such that each may be elutedseparately or selectively without significant or substantialcontamination of the other).

In some implementations, the migration of separated bands of analytesand serial elution of isolated bands of one or more analytes can beconducted while still maintaining separation of the analytes byproviding appropriate counterbalancing force to prevent mixing of theseparated analytes. For example, a counter balancing force of negativepressure (via vacuum source) can be used to counter the effects ofgravity on a vertically oriented capillary during elution of separatedanalytes.

In some implementations, the visualization and/or detection of analytescan be conducted in a real-time or semi-real-time manner such that theseparated analytes and their relative localization (e.g., localizationof a peak concentration of each separated analyte in the fluid path) canbe monitored as the separated analytes are made to migrate towards thedistal end of the capillary. In some implementations, the apparatus canbe manipulated such that a movement of a sample plate including aplurality of collection wells can be coordinated based on the relativelocalization of analytes (e.g., relative location of peak concentrationsof each separated analyte) and/or a rate of migration of each analyte(e.g., rate of migration of a peak concentration of each analyte). Forexample, the sample plate can include multiple collection wells and thecapillary can be moved between collection wells eluting a fraction ofthe sample (e.g., one or more separated analytes) into each collectionwell. In some embodiments, each collection well can contain a chemicalmobilizer (e.g., the same or different chemical mobilizers), and thecapillary can be moved from collection well to collection well. While ina collection well a voltage can be applied until a fraction of thesample is eluted. Once the fraction of the sample is eluted (e.g., asdetermined by continuous monitoring of the capillary), the capillary canbe moved to another collection well, where a subsequent fraction of thesample can be eluted. In addition, or as an alternative to chemicalmobilization, a fraction of the sample can be eluted into eachcollection well by applying a pressure to the capillary.

A Fractionation System

FIG. 1 is a schematic illustration of a portion of a fractionationsystem 100 (also referred to herein as “the system”) configured toperform separation, detection, and/or fractionation of one or moreanalytes in a sample (e.g., based on molecular weight and/or isoelectricpoint) according to an embodiment.

Embodiments of the fractionation system 100 described herein can be usedto facilitate separation of one or more analytes in a single system,visualization and/or detection of analytes within a sample before,during, and/or after the separation, and fractionation of one or moreseparated analytes based on the separation and visualization anddetection. Embodiments described herein can provide the functionality ofpipettes and microfluidic paths, thereby enabling the separation,analysis, and/or fractionation of very small volume samples. Suchapparatus and/or systems can include any suitable device, mechanism,assembly, subassembly, electronic device, actuator, and/or the like thatcan enable the apparatus and/or system to, for example, separate,visualize, detect, and/or fractionate any suitable target analytes.

The system 100 includes a housing 101, a probe system 102, a cartridgeretainer 103 configured to receive and/or secure a capillary cartridge104, a sample plate assembly 107, and an electronic system 108. Whilenot shown in FIG. 1 , the electronic system 108 can include a processor,a memory, a communicator, and/or a power source. The electronic system108 can be configured to permit communications with external computedevices using any suitable mode of communication, for example, toreceive/transmit data and/or instructions. In some embodiments, thesystem 100 can be configured such that the electronic system 108includes any suitable system or assembly with a power source, aprocessor, and a memory that can be configured and/or otherwiseprogrammed to perform one or more processes (e.g., hardware moduleand/or software module stored in the memory and executed in theprocessor) associated with performing at least a semi-automaticelectrophoretic separation. Similarly, the system 100 can include anysuitable fluid flow system or assembly that defines one or more fluidflow paths configured to receive a fluid such as, for example, a sample,one or more reagents, and/or the like, which can flow through the system100 as described in further detail herein with reference to specificembodiments.

The housing 101 of the system 100 can be any suitable shape, size, orconfiguration and can be arranged to at least partially enclose or atleast partially house any suitable component of the system 100. Forexample, the housing 101 can at least partially enclose the probe system102, the sample plate assembly 107, the capillary cartridge retainer103, and the electronic system 108. Although not shown in FIG. 1 , insome embodiments, the housing 101 can be configured to form one or moreportions, chambers, inner volumes, etc. that are configured to allow atleast some of the components of the system 100 to be disposed therein.In some embodiments, the housing 100 can include a door configured toprovide access to the inner volume defined thereby. For example, a usercan open the door of the housing 100 to position a capillary cartridge104 within the capillary cartridge retainer 103, as described in furtherdetail herein. In some embodiments, at least a portion of the housing101 can be light tight such that no substantial quantity of light leaksthrough the housing into a chamber defined by the housing. In someembodiments, the housing 101 can define at least one climate- controlledchamber. Similarly stated, the system 100 can be operable to maintain achamber of the housing at a constant and/or preset temperature,humidity, and/or other environmental parameter (e.g., illumination,etc.).

The probe system 102 of the system 100 can be fixedly disposed withinthe housing 101. In some embodiments, the probe system 102 can bedisposed in a predetermined and fixed position relative to the cartridgeretainer 103 and/or one or more components in association with thecartridge retainer 103 or the cartridge 104 (e.g., a viewing window (notshown) defined on the cartridge, the capillary 106, etc.,). For example,the probe system 102 can be arranged within the housing 101 such thatpredetermined portions of the probe system 102 are aligned with and/orotherwise disposed in a desired position relative to predeterminedportions of the cartridge retainer 103. In some embodiments, the probesystem 102 and/or cartridge retainer 103 can include any suitableadjustment mechanism or the like to ensure a desired alignment betweenthe probe system 102 and the cartridge retainer 103.

The probe system 102 can include any suitable device, mechanism, and/orassembly that is configured to capture and/or detect digital or analogdata (e.g., images) of, for example, an analyte and/or standard and/orto detect a signal emitted by the analyte and/or standard (e.g., in asample held in the capillary 106) such that any suitable analyses may beperformed using the signal (e.g., analyses conducted by the electronicsystem 108). In some embodiments, the probe system 102 can include oneor more emitters and one or more detectors (not shown in FIG. 1 ).

The emitters included in the probe system 102 can be any suitabledevice, member, mechanism, assembly, and/or the like that is configuredto release energy (e.g., heat, photons, radiation, etc.). For example,in some embodiments, the emitters can include LEDs, arrays of LEDs, adeuterium lamp, a laser, an incandescent light source, a fluorescentlight source, or any other suitable light source. The emitter, in someembodiments, can be optically coupled to the cartridge retainer 103, thecartridge 104, and/or the capillary 106 via one or more lenses, mirrors,prisms, fiber optics, and/or the like. The emitter(s) can be poweredand/or excited to emit light at, for example, a predetermined wavelengthand/or range of wavelengths. In some embodiments, the probe system 102can include one or more mirrors, lenses, filters and/or the likeconfigured to direct, focus, and/or convert the wavelength of photonsemitted by the emitter(s). For example, the probe system 102 can includeany suitable lens and/or filter (e.g., a TAMRA filter) that isassociated with chemiluminescence, fluorescence (e.g., nativefluorescence, fluorescence of label moieties, etc.), absorbance, and/orthe like. In some embodiments, the probe system 102 can include asequence of emitters, for example, a grid array of fiber optic outputs,LEDs, or the like (e.g., a column of light). In some embodiments, theprobe system 102 can be configured such that the one or more emitterscan be used to convey energy (e.g., excitation energy) to a sample viaone or more apertures, filters, blockers, reflectors and/or refractors,etc. The energy conveyed can be configured to interact with at least aportion of a sample contained within the capillary 106 of the cartridge104 when the cartridge 104 is retained by the cartridge retainer 103.

The detectors included in the probe system 102 can be any device thatcan receive or acquire a signal emitted or associated with a portion ofa sample in the capillary 106 and convey data or information associatedwith the acquired signal to a processor (e.g., a processor included inthe electronic system 108). In some embodiments, the detectors can beconfigured to receive signals in one form that can be transduced toanother form or to data that can be transmitted to the processor. Forexample, the detectors can include any suitable digital or analogdetectors that can capture a signal in the form of light emitted by aportion of the sample and transduce the captured signal into data (e.g.,digital data conveying information related to intensity, wavelength,quality, duration of emission, etc.) that can be used to performsuitable analyses of the portion of the sample from which the signal wasreceived. As an example, in some embodiments, one or more detectors canbe and/or can include a photodiode, an array of photodiodes, aphotomultiplier tube (PMT), a charged coupled device (CCD) array, and/orthe like. The detectors can be used to capture an image and/or signalassociated with the analyte and/or standard within a sample. In someembodiments, the detectors can be operable to capture images and/orsignals emitted from the analyte and/or standard periodically and/orcontinuously. In some embodiments, the detectors can be operable tomonitor the analyte and/or standard, in real time or substantially inreal time, which can allow a user to rapidly determine whether ananalyte is present in the sample, a rate of migration of a portion ofthe sample (e.g., during elution), an amount or activity of the analyte,a molecular weight of the analyte, and/or the like.

In some embodiments, a detector can be used during a fractionation todetect and/or image, substantially in real time, a flow of a samplethrough the capillary 106 of the cartridge 104 (in this instance,configured for use in fractionation based on isoelectric focusing andassociated analyses) when the cartridge 104 is retained by the cartridgeretainer 103. In some embodiments, a detector can be used during and/orafter isoelectric focusing. For example, the detector can be used todetect the separation of analytes substantially in real time as analytesseparate and focus and/or after analytes have been focused and,optionally, migrated towards an end of the capillary to be fractionatedout in isolation. Similarly stated, the detector can detect a signal(e.g., fluorescence, absorbance, etc.) associated with one or moreanalytes included in a sample that has been separated and/or is in theprocess of being eluted from within the capillary 106 of the cartridge104 (in this instance, configured for use in isoelectric focusing) whenthe cartridge 104 is retained by the cartridge retainer 103. As describepreviously, the probe system 102 can be operably coupled to any suitableelectrical or electronic circuit included in the electronic system 108and/or associated with a remote device. The probe system 102 can beconfigured to send and/or receive signals from a processor and/or thelike (e.g., the probe system 102 can send one or more signals to theprocessor or the like in the electronic system 108 to cause dataassociated with the captured images and/or detected signals to bestored, for example, in a memory or database). The probe system 102 caninclude a single detector or multiple detectors (e.g., more than two)configured to detect a portion of energy (e.g., light of specifiedwavelength range) produced by the emitters and/or interacted by aportion of the sample (e.g., fluorescence from a separated analyte in asample held in capillary 106).

As described above, the system 100 is configured to receive a capillarycartridge 104 (also referred to herein as “cartridge”). In someembodiments, the system 100 is configured to receive a cartridge 104including one or more capillaries 106 and to expose at least a portionof the cartridge 104 to negative pressure differential (e.g., producedby a vacuum source) operable to draw a volume of fluid (e.g., one ormore reagents, samples, buffers, washes, detectors, analytes,ampholytes, and/or the like) from one or more wells or trays included inthe apparatus and/or system into the capillary(ies) 106 of the cartridge104.

In some embodiments, the system 100 is configured to receive thecartridge 104 including capillaries 106 and to expose at least a portionof the cartridge 104 to a positive pressure differential (e.g., producedby a pressure source) operable to inject or eject a volume of fluid(e.g., one or more reagents, samples, buffers, washes, detectors,analytes, ampholytes, and/or the like) from the capillary 106 to one ormore wells or trays.

In some embodiments, the system 100 is configured to receive thecartridge 104 including one or more capillaries 106 and to expose atleast a portion of the cartridge 104 to a source of applied voltage or asource of electric current that can be operable to draw a volume offluid via electrokinetic injection (e.g., one or more reagents, samples,buffers, washes, detectors, analytes, ampholytes, and/or the like) fromone or more wells or trays into the capillary 106. In some embodiments,the system 100 can be configured to expose at least a portion of thecartridge 104 to a source of applied voltage or a source of electriccurrent that can be operable to inject a volume of fluid viaelectrokinetic injection (e.g., one or more reagents, samples, buffers,washes, detectors, analytes, ampholytes, and/or the like) from thecapillary 106 to one or more wells or trays. Electrokinetic injectioncan be injection of a substance (injectate) by applying a voltage or acurrent via a substance in a capillary 106. An amount of the substanceinjected into a well or vial can depend on a mobility of componentsincluded in the substance, a diameter of the capillary 106, the appliedelectric field and/or the injection time (e.g., time of application ofthe voltage).

The cartridge 104 can include at least a body portion that is fixedlycoupled to at least one capillary 106. The cartridge 104 can be anysuitable shape, size, or configuration.

The capillary cartridge retainer 103 (also referred to herein as“cartridge retainer”) is fixedly disposed within the housing 101. Forexample, in some embodiments, the cartridge retainer 103 can be coupledto a frame or the like that maintains the cartridge retainer 103 in asubstantially fixed position within the housing 101. In someembodiments, the cartridge retainer 103 can also be coupled to and/orotherwise disposed in a fixed position relative to the analysis system102, as described in further detail herein.

The cartridge retainer 103 can be any suitable shape, size, orconfiguration. For example, the cartridge retainer 103 can include, forexample, a set of sidewalls that define an inner volume configured toreceive at least a portion of a capillary cartridge 104 (also referredto herein as “cartridge”). More particularly, the cartridge retainer 103can have or define a substantially C-shaped cross-section with at leastone side of the cartridge retainer 103 being substantially open. A usercan insert the cartridge 104 through the substantially open side of thecartridge retainer 103 to position at least a portion of the cartridge104 within the inner volume. In some embodiments, the cartridge retainer103 can include a latch mechanism suitable to form a friction fit, asnap fit, a threaded coupling, and/or the like with at least a portionof the cartridge 104 to couple the cartridge 104 to the cartridgeretainer 103. In other words, the cartridge retainer 103 at leasttemporarily couples to the cartridge 104 when the portion of thecartridge 104 is inserted into the inner volume to maintain thecartridge 104 in a substantially fixed position relative to thecartridge retainer 103.

The cartridge retainer 103 can be configured to receive the cartridge104 in a predetermined orientation (e.g., only one orientation or way).Although not shown in FIG. 1 , the cartridge retainer 103 can includeany suitable alignment feature or sensor configured to engage and/orsense a portion of the cartridge 104 as the cartridge 104 is positionedwithin the cartridge retainer 103. More particularly, the cartridgeretainer 103 can include, for example, any number of features (e.g.,protrusions, openings, grooves, etc.), assemblies, mechanisms, sensors,and/or the like, each of which engage and/or sense a portion of thecartridge 104 to ensure the cartridge 104 is retained within thecartridge retainer 103 at a desired position and/or in a desiredorientation.

The cartridge 104 is configured to receive and house a capillary 106 ina specified orientation such that when the cartridge 104 is engaged withthe cartridge retainer 103 the capillary 106 can be engaged with one ormore components of the system 100 to enable forming a fluid path thatcan be used to manipulate a sample (e.g., draw the sample, separateanalytes in the sample, analyze one or more constituents of the sample,fractionate one or more analytes, etc.)

The capillary 106 is configured to be placed in fluid communication withone or more fluid reservoirs (e.g., disposed in the cartridge bodyand/or disposed in or defined by a reagent tray or the like). In someembodiments, the one or more fluid reservoirs can be wells or the likecontaining a fluid with constituents having any of the chemistriesdescribed above. In some embodiments, the one or more fluid reservoirscan be wells (e.g., sample collection wells) having solutions forseparation and/or fractionation of analytes including running buffers(e.g., acid or base solutions), chemical mobilizers, etc. as describedherein.

The capillary 106 of the cartridge 104 defines a lumen that receives atleast a portion of a sample, solution, reagent, analyte, and/or anyother suitable fluid or gel. In some embodiments, the capillary 106 caninclude a separation matrix configured to support generation of a pHgradient and/or separation of analytes (e.g., via isoelectric focusing).In some embodiments, the capillary 106 of a cartridge 104 can be anelongate member having a rounded or circular cross-sectional shape or apolygonal cross-sectional shape (e.g., trapezoidal, rectangular, square,pentagonal, octagonal, etc.). In some embodiments, the shape and/or sizeof the lumen defined by the capillary 106 can be based at least in parton the sample, the sample volume, and/or the type of analysis (e.g.,with an inner diameter of about 10 micrometers or “microns” (μm) toabout 1000 μm). For example, a capillary 106 having a relatively smallinner diameter can be associated with and/or otherwise used forrelatively low sample volumes, which can be suitable for expensivesamples or reagents. Conversely, a capillary 106 defining a relativelylarger inner diameter can be associated with and/or otherwise used forrelatively high sample volumes, which in some instances, can result inimproved signal detection or the like. In other embodiments, the innerdiameter can be based at least in part on the analysis to be performed(e.g., molecular weight-based separation, isoelectric focusing, etc.).In some embodiments, capillaries with multiple segments having differentinner diameters (including transition segments with variable innerdiameter) can be used, as described herein.

The capillary 106 can be any suitable shape, size, volume, orconfiguration and can be formed from any suitable material (e.g., glass,plastic, silicon, fused silica, gel, metal, carbon nanotubes, PYREX™(amorphous glass), and/or the like) that allows a liquid and/ordissolved molecules to flow through the lumen. The capillary 106 canhave any suitable length and any suitable inner diameter and a suitableouter diameter. For example, in some embodiments, the capillary 106 canhave a length of approximately 50 to 120 mm (e.g., a length as small as5 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 60 mm 70 mm, 80 mm, 90 mm, 100mm, 110 mm, 120 mm, or any suitable length therebetween). In someembodiments, the capillary 106 can have a length of approximately 100 mmto 1000 mm (e.g., a length as small as 100 mm, 200 mm, 300 mm, 400 mm,500 mm, 600 mm 700 mm, 800 mm, 900 mm, 1000 mm, 1100 mm, or any suitablelength therebetween). In some embodiments, the capillary 106 can have asingle constant inner diameter and/or outer diameter. For example, insome embodiments the capillary can have an inner diameter ofapproximately 320 to 530 μm (e.g., an inner diameter of 320 μm, 420 μm,530 μm, or any suitable inner diameter therebetween.

In some embodiments, the length of the capillary can be based at leastin part on factors such as sample size or volume and the extent ofsample separation when resolving the analyte or analytes of interest(e.g., between about 2 centimeters (cm) and about 20 cm), where a longercapillary can result in increased separation of samples, which in turn,can improve resolution of complex mixtures and/or mixtures having a lowabundance of analytes. In some embodiments, the capillary 106 can haveany suitable diameter which can be based at least in part on factorssuch as sample size or volume, an extent of sample separation whenresolving the analyte or analytes of interest, a speed of separation, aspeed of migration for elution or fractionation of analytes in a sample(e.g., between 1 mm/min and 5 mm/min), where a capillary with a largerdiameter can result in larger volumes and/or increased speed ofmigration of separated analytes in the samples, which in turn, canimprove or increase a fractionated amount of a separated analyte, and/ora speed of fractionation of the analytes from a complex mixtures and/ormixtures in the sample. The diameter of the capillary can be based atleast in part on factors such as sample size or volume, the extent ofsample separation desired for an analyte or analytes of interest, and/ora speed of fractionation.

In some embodiments, the capillary 106 can include two or more portionseach portion associated with a specified length and location and eachportion having a specified variable inner diameter and/or outerdiameter, as described in further detail in following sections. In someembodiments, the capillary 106 can be made as a unibody element. In someembodiments, the capillary 106 can be made by joining two or moreportions together.

In some embodiments, the cartridge retainer 103 can include and/orotherwise can be coupled to any suitable assembly, mechanism, device,and/or the like configured to engage the cartridge 104 to control, forexample, a flow of fluid through at least a portion of the cartridge104. For example, in some embodiments, the cartridge retainer 103 caninclude and/or can be coupled to a vacuum source or assembly (not shownin FIG. 1 ). The vacuum source or assembly can be configured to bebrought in fluidic connection with the cartridge 104 via suitableconnection (e.g., tubing, port, and/or the like) and the vacuum sourceor assembly can be configured to produce a negative pressure within avolume of the cartridge 104 such as, for example, within a lumen of acapillary 106 or a portion of a fluidic path defined in the cartridgeand/or the like. Said in another way, the vacuum source can befluidically coupled to a portion of the cartridge retainer 103 via aport or a tubing or the like that places the vacuum source in fluidcommunication with the lumen of the capillary 106 in the cartridge 104when the cartridge 104 is retained by the cartridge retainer 103.

In some instances, the vacuum source can be a pressure source capable ofapplying positive and/or negative pressure differential. For example,the pressure source can apply a negative pressure differential to draw asample or substance from a sample well into the capillary 106. Asanother example, the pressure source can apply a positive pressuredifferential to inject or elute a portion of the sample or substance(e.g., a separated analyte or set of analytes) from the capillary 106and into a collection well. In some embodiments, the pressure source canbe activated and/or controlled (e.g., by a manual switch or controlledand/or by an electrical switch or controller included in an electricalcircuit and controlled by a processor) to produce and/or modulate anegative pressure of a desired magnitude within the cartridge 104, asdescribed in detail below with reference to specific embodiments. Insome embodiments, the cartridge retainer 103 can include a device ormechanism configured to engage the cartridge 104 to selectively limit abulk flow of fluid through a portion of the cartridge 104. For example,the cartridge retainer 103 can include an actuator or the like that isselectively placed in contact with a pinch valve or the like included inthe cartridge 104 to limit and/or substantially prevent a bulk flow offluid through, for example, a lumen of a capillary included in thecapillary cartridge 104.

In some embodiments, the cartridge 104 can include one or more runningbuffer reservoirs 105 that can be configured to hold one or more runningbuffers that can be used to probe, separate, analyze, and/or fractionateone or more analytes in a sample held in the capillary 106. As anexample, a first running buffer held in the running buffer reservoir 105can include a buffer having a first pH and/or having a specifiedconcentration of desired ions. For example, in some embodiments, therunning buffer reservoir 105 can hold a specified quantity of an acidwith a desired pH. The capillary 106 can be positioned such that a lumenof the capillary 106 at a first end or a proximal end of the capillary106 is fluidically and/or ionically coupled with the running buffer heldin the running buffer reservoir 105. The acid and/or its pH may beselected such that one or more analytes included in a sample, that isdrawn into the capillary 106, can be separated using techniques likeisoelectric focusing by taking advantage of the pH of the runningbuffer. That is, in some embodiments, the sample in the capillary can beionically coupled via a first end (e.g., a proximal end) to a firstrunning buffer in the running buffer reservoir and having a first pH.The sample in the capillary can be ionically coupled via a second end(e.g., a distal end) to a second running buffer in a well and having asecond pH different than the first pH. The well holding the secondrunning buffer can, for example, be in the sample plate held by thesample plate assembly 107, and the distal end of the capillary may beconfigured to be dipped into the well by the system 100. The firstrunning buffer at the first pH and the second running buffer at thesecond pH can induce a pH gradient to be established along the capillarylumen and through the sample. The pH gradient can cause one or moreanalytes in the sample to separate and/or migrate along the gradient tobe positioned and/or accumulated at their respective equilibrium pHbased on the ionic balance of the respective analytes.

Following separation and/or focusing of the one or more analytes in thesample along the pH gradient the one or more analytes can be mobilizedand eluted using any suitable process including hydrodynamicmobilization and/or chemical mobilization. Hydrodynamic mobilization canrefer to a mobilization of analytes due to movement of fluids (e.g., dueto a flow driven by gravitational forces acting on fluids in avertically oriented capillary, a flow driven by a pressure differential,and/or the like). In some instances, the application of electric fieldcan be continued during hydrodynamic mobilization, which can help tomaintain separation between the analytes. Chemical mobilization canrefer to a mobilization of analytes by a process in which an anolyte(electrolyte on the anode side of the capillary 106) or a catholyte(electrolyte on the cathode side of the capillary) can be replaced byanother electrolyte with a high ionic strength and/or a different pHcompared to the running buffers associated with the anode side orcathode side of the capillary 106. The electrolyte with the high ionicstrength and/or different pH can be introduced via a chemical mobilizer(also referred to herein as an elution buffer) in conjunction with anapplied electric field between a first running buffer and the chemicalmobilizer via the capillary 106.

A chemical mobilizer can be a buffer that provides ions that can migrateinto the capillary 106 and disrupt the pH gradient in the capillary 106to again impart a charge to the separated analytes that may have beenrendered neutralized and stable. When a one end (e.g., a distal end) ofthe capillary 106 is disposed in a chemical mobilizer the electrolytecan be introduced into the capillary 106 and can disrupt the pHgradient. Ampholytic analytes in the now-disrupted pH gradient cantherefore experience a force causing them to migrate and/or be eluted.In some embodiments, the cartridge 104 can be configured such that thefirst running buffer in the running buffer reservoir 105 can be used toelute and/or fractionate one or more analytes in a sample held in thecapillary 106 in conjunction with a chemical mobilizer. As an example,the first running buffer held in the running buffer reservoir 105 caninclude a buffer having a first pH and/or having a specifiedconcentration of desired ions. For example, in some embodiments, therunning buffer reservoir 105 can hold a specified quantity of an acidwith a desired pH. A collection well can include a chemical mobilizerwith a buffer having a third pH and/or having a third specifiedconcentration of a desired set of ions, for example a specified quantityof base with a desired pH.

In some implementations, a single chemical mobilizer can be chosen toelute separated analytes in a capillary 106. One or more wells holdingthe chemical mobilizer can, for example, be in the sample plate held bythe sample plate assembly 107, and the distal end of the capillary maybe configured to be disposed into the well holding the chemicalmobilizer by the system 100. The chemical mobilizer can induce elutionof the separated analytes along the pH gradient generated during theseparation phase (e.g., established using the second running buffer)such that analytes focused in a specified portion of the pH gradient canbe collected in the collection well. In some implementations, multiplewells can contain the chemical mobilizer such that the distal end of thecapillary 106 can be moved from collection well to collection well, anda specified portion of the separated one or more analytes can becollected in each (or at least a subset) of the wells. Movement of thecapillary 106 from collection well to collection well can be coordinated(e.g., with real-time imaging of the column) such that separatedanalytes with slightly different pI values can be tracked and/orselectively eluted and collected as fractions in separate collectionwell via chemical mobilization.

Some of the properties of a chemical mobilizer/elution buffer to beconsidered when choosing chemical mobilizers for mobilizing analytesinclude: (1) compatibility with mass spectrometry (e.g., a volatility ofchemical mobilizers can help remove the chemical mobilizer componentsduring electrospray during a mass spectroscopy analysis followingfractionation and collection), (2) a pH that permits stable storage ofthe analytes (e.g., proteins) that may remain in the chemical mobilierafter elution (e.g., a pH range of 4 to 9, in some implementations, a pHrange of 5 to 8), (3) sufficient buffer capacity to support the currentduring chemical mobilization. In some implementations, a sufficientbuffer capacity can be achieved by increasing a concentration of ions inthe chemical mobilizer/elution buffer. Some example chemical mobilizersinclude NaCl, Acetic Acid, Acetate salt, Formic Acid, Phosphate Salt,Phosphoric Acid, Ammonium Acetate, and Formate. Ammonium Acetate is anexample chemical mobilizer that can have a pH 6.7 and can be used tomobilize all analytes (e.g., proteins) in a sample. Chemical mobilizercan be chosen at a suitable concentration based on desired mobilizationand/or elution parameters (e.g., speed, precision, etc.) As an example,NaCl can be chosen as a chemical mobilizer at a concentration of about10 to 200 mM, acetic acid can be used at a concentrations from about 20mM to 200 mM, acetate salt (e.g., ammonium) can be used at aconcentration from about 1 mM to 20 mM, formic acid can be used at aconcentration from about 0.01% to 1%, phosphate salt can be used at aconcentration from about 0.5 mM to 20 mM, formate can be used at aconcentration from about 0.5 mM to 20 mM.

The cartridge retainer 103 can also include any number of electricalcontacts (not shown in FIG. 1 ) or the like configured to electricallycouple a portion the cartridge 104 to an electrical or electronicassembly included in the system 100. For example, in some embodiments,the cartridge retainer 103, the cartridge 104, and/or the capillary 106can include one or more electrically conductive contact members, clips,surfaces, etc. that are placed in contact with an associatedelectrically conductive contact member, clip, surface, etc. of thecartridge 104 when the cartridge 104 is retained therein. In someembodiments, at least a portion the cartridge 104 can be formed from anelectrically conductive material such as stainless steel, electricallyconductive plastic or the like. Thus, when the cartridge 104 is retainedin a desired position within the cartridge retainer 103 the one or moreelectrically conductive members or the like of the cartridge retainer103 contact one or more predetermined electrically conductive portionsof the cartridge 104, which in turn, places the cartridge 104 inelectrical or electronic communication with the electrical and/orelectronic assembly included in the system 100. By way of example, thecartridge retainer 103 can electrically connect a conductive capillaryof the cartridge 104 and/or a conductive fluid within a lumen of acapillary to the electrical and/or electronic assembly of the system100, as described in further detail herein.

In some embodiments, at least a part of the cartridge 104 and/or thecapillary 106 can be electrically conductive. The electricallyconductive part of the cartridge 104 and/or the capillary 106 can beformed from any suitable electrically conductive material. For example,the electrically conductive part can be formed from a metal includingcopper, platinum, stainless steel, electrically conductive microplateplastic, carbon-infused plastic, an electrically conductive polymer,and/or any other suitable material. As an example, in some embodiments,the cartridge 104 can include a capillary 106 with a metal tip at thedistal end of the capillary that is electrically conductive. When thecapillary is dipped into a solution (e.g., in a collection well or areagent well in a sample plate) the metal tip can be disposed in thesolution and can be used to provide an electrical connectivity betweenan external source of electrical power and the solution and /or thelumen of the capillary. The metal tip can be used to apply a voltagethrough the solution that is occupying the lumen of the capillary andthat optionally contains a pH gradient between ionically coupled runningbuffers (e.g., disposed on opposite sides of the capillary). Said inanother way, the metal tip can be used to provide a voltage across thelength of the capillary 106 and between solutions and/or buffers thatare coupled to the proximal and distal ends of the capillary 106,respectively, such that analytes in a sample held in the lumen of thecapillary 106 can be separated and/or fractionated by applying avoltage.

In some embodiments, the cartridge 104 can include one or moreelectrical leads or points of electrical connectivity that areconfigured such that the first running buffer and/or the second runningbuffer that is ionically coupled to the sample in the capillary can eachbe also electrically coupled to a power source (e.g., a power sourceincluded in the electronic system 108). In some embodiments, asdescribed in further detail in the following sections, the first runningbuffer can be electrically coupled to a first electrical leadelectrically coupled to the running buffer reservoir and a voltagesource, and the second running buffer (e.g., in a well in a sample plateheld by the sample plate assembly 107) can be electrically coupled to asecond lead positioned at the distal tip on the capillary 106 holdingthe sample based on the distal end of the capillary being dipped intothe second running buffer. The second lead positioned at the distal tipof the capillary 106 can be configured to be electrically coupled to thevoltage source such that the system 100 can provide apply a specifiedvoltage across the first lead and the second lead, thereby applying thevoltage between the first running buffer and the second running buffervia the sample held in the lumen of the capillary 106. In someinstances, the voltage applied can be selected to induce isoelectricfocusing of the components included in the sample such that one or moreanalytes and/or standards included in the sample can separate and/ormigrate to their respective isoelectric points (pI) (i.e., the pH atwhich each analyte has no net charge and therefore is at equilibriumwith no more net movement along the pH gradient) along the length of thecapillary based on their respective ionic balance with respect to the pHgradient generated between the first running buffer and the secondrunning buffer.

As described previously, the capillary 106 can be of any suitable shape,size, or configuration and can be arranged to be received by thecartridge 104 and to have a lumen that can be made continuous with afluidic path that is defined in the cartridge 104 (e.g., a lumen that isconfigured to be arranged in fluidic connection with a tubing that iscoupled to a vacuum source, to a first running buffer in a runningbuffer reservoir, to a sample well in a sample well plate, to a secondrunning buffer in a well included in a sample well plate, etc.

Some embodiments described herein relate to capillary-containingcartridges suitable for use with capillary electrophoresis instruments,such as Maurice by ProteinSimple®. U.S. Pat. No. 10,794,860, issued onOct. 6, 2020 and entitled “Systems and Method for CapillaryElectrophoresis, Isoelectric Point, and Molecular Weigh Analysis,” theentire disclosure of which is hereby incorporated by reference, includesadditional disclosure of a suitable capillary electrophoresis instrumentand cartridges suitable for capillary electrophoresis. Embodimentsdescribed herein generally relate to cartridges that include a capillary106 that can serve or that includes portions that can serve to transfersolutions, hold a sample including analytes and/or standards, support aseparation of one or more analytes/standards in the sample, and eluteone or more separated analytes via fractionation. The capillary 106 canbe configured to transfer a buffer or maintain fluidic contact with oneor more buffer reservoirs such that the capillary 106 can be to be incontact with a buffer reservoir(s). The capillary 106 can be configuredto transfer a sample and/or receive the sample into a lumen of thecapillary 106 such that the contents in a lumen of the capillary can besubjected to an applied voltage to form a pH gradient.

In some embodiments, the cartridge 104 and the capillary 106 can beconfigured such that the capillary 106 is in a substantially verticalorientation with respect to gravity with the distal end protruding awayfrom the cartridge 104 such that a portion of the distal end can beremovably disposed in any suitable well, vial, reservoir, and/or thelike that can be introduced at the correct position with reference tothe capillary 106 and/or the cartridge 104. In some embodiments, theprotruding end (e.g., a distal end) of the capillary 106 can beconfigured to be disposed in sample reservoir in a sample well plateand/or a buffer reservoir in collection wells (e.g., in a sample wellplate).

In some embodiments, the proximal end of the capillary 106 can beconfigured to be coupled to a suitable mechanism that can be used toload a sample into the lumen of the capillary 106. For example, during aloading step suction applied through a sheath interface or a tubingcoupled to the proximal end of the capillary 106 via the vacuum sourcecan provide a negative pressure to draw a sample or buffer from a sampleor buffer reservoir and bring the sample or buffer into the lumen of thecapillary 106.

The capillary 106 can be configured for separation of analytes includedin the sample, for example via isoelectric focusing when an electricpotential (i.e., voltage) is applied across the proximal and distal endsof the capillary 106. The electric potential can be applied across thelumen of a substantially vertically oriented capillary 106 via a firstor top running buffer reservoir and a second or bottom running bufferreservoir. In some embodiments, the capillary 106 can be configured suchthat one end of the capillary 106 (e.g., a proximal end) can be disposedin a first running buffer reservoir included in the cartridge 104. Theother end protruding from the cartridge 104 can be configured to bedisposed in a second running buffer reservoir in a sample well includedin a sample well plate. Said in another way, the top running bufferreservoir can be disposed in the cartridge 104, and a top end of thecapillary 106 can be disposed in the top running buffer reservoir. Abottom of the capillary 106 can be disposed in a bottom running bufferreservoir, which can be disposed in a sample plate that is a portion ofor accessed by the system 100. Similarly stated, the capillary 106 canextend from the cartridge and be “dipped” into a bottom running bufferreservoir.

In some embodiments, one or both ends of the capillary 106 can beconfigured to include a portion of a porous membrane (not shown in FIG.1 ) having a predefined pore size and covering an access to the lumen ofthe capillary 106. Some embodiments can include membranes on both endsof the capillary used to separate the analytes. Some embodiments caninclude a membrane on just one end of the capillary 106. For example,the proximal (e.g., top) end of the capillary 106 that is disposed in afirst running buffer held in a first running buffer reservoir caninclude a porous membrane that reduces movement of the first runningbuffer into the capillary 106, for example due to a gravitational and/orcapillary forces. The porous membrane(s) can serve as a hydrodynamicbarrier preventing the first running buffer (e.g., acid) and/or thesecond running buffer (e.g., base) from entering the capillary duringseparation. The ions such as H⁺ and OH⁻ can, however, freely transportthrough the membrane(s) under electric field allowing the separation(e.g., via isoelectric focusing process) to occur. In addition to themembranes, some embodiments can include one or more valves located at orclose to one or more ends of the capillary 106 (e.g., a loading end ofthe capillary used to load sample, an elution end of the capillary usedto elute fractions). The valve at a loading end/elution end can beconfigured to be opened during sample loading and/or fraction elutionand to be closed during the separation phase to minimize hydrodynamicflow during separation. In some embodiments, the porous membrane may beconfigured to be removed during elution (e.g., using a suitableactuation).

In some embodiments, a porous membrane at a distal (e.g., bottom)portion of the capillary, through which sample is injected and/orfractions eluted, can have a predefined selectivity to reduceinfiltration of the first running buffer into the lumen of the capillary106 while allowing analytes and/or protons and/or hydroxyl ions to passtherethrough under an applied electric field. In some embodiments, theporous membrane can be in the form of a tubing coupled to one end (e.g.,the proximal end) of the capillary, and also referred to as “membranetubing”. Such a membrane tubing defines a lumen through which fluid canflow hydrodynamically when a pressure difference exists between the twoends of the membrane tubing. In contrast, the porous wall that enclosesthe lumen can permit transport of ions (e.g., by electrophoresis ordiffusion) while substantially restricting hydrodynamic flow. In someembodiments, the membrane tubing can be configured to connect a vacuumsource to the capillary 106 (e.g., at the proximal end of the capillary106) and can be in contact with and/or submerged in the running bufferreservoir. The porous membrane or membrane tubing can have a molecularweight cut-off (MWCO) of approximately 10 kDa to 500 kDa in someembodiments. Some such embodiments, the porous membrane can beconfigured to be used with a cartridge 104 with a capillary 106 that hasa single inner diameter of about 320-530 μm with a length of about60-120 mm. Some such embodiments can be configured to increase a sampleloading capacity while at the same time maintaining an advantage of realtime monitoring of progress of separation of analytes, for example viaisoelectric focusing. In some embodiments, the porous membrane can beselected to enable effective mobilization speed during chemicalmobilization and elution of fractions of the sample. The porous membranecan be selected to reduce a disruption of a pH gradient generated duringseparation at the stage of mobilization and elution of fractions.

In some embodiments, the selection of the porous membrane can be basedon a size location, and/or orientation of the running buffer reservoir(e.g., 500 ∥l-4000 μl), a volume associated with the membrane tubing,and/or an inner and/or outer diameter of the capillary 106. For example,the volume associated with the membrane tubing can be a portion oftubing of approximately 300 μm to 700 um inner diameter, and a length ofapproximately 0.5 mm to 2 mm. In some instances, the porous membrane,the running buffer reservoir (and the cartridge associated with arunning buffer reservoir), and the capillary can be selected to suitablymatch each other to achieve a desired separation, mobilization, and/orelution of analytes (e.g., a target efficiency of separation, a targetspeed of mobilization, etc.).

A size, location, and/or orientation of the running buffer reservoir canaffect a hydrodynamic flow associated with contents of the lumen of thecapillary 106, as does the porous membrane (e.g., pore size of themembrane). For example, a volume and/or size of the running bufferreservoir that may be located and/or oriented at a vertically elevatedposition with respect to a capillary 106 can impart a hydrodynamic flowbased on gravitational forces acting on the running buffer released fromthe running buffer reservoir. The pore size of the porous membrane cannot only affect the degree of hydrodynamic flow but also the efficiencyof ion-exchange (i.e., level of electric current under applied voltage).In some embodiments, the running buffer reservoir can be configured tohold a first running buffer which for example can be an acid. Therunning buffer reservoir can thus serve as a supplier of protons duringthe separation and mobilization process. The larger the size or volumeof the sample to be separated, which can depend inner diameter of thecapillary 106, the larger may be the need for protons. Thus, the innerdiameter of the capillary 106 can affect the sample size, which candetermine a size of running buffer reservoir that may be used to supplya suitable number of protons that can be supplied.

When a sample is introduced into the cartridge 104, the sample candistribute in both the lumen of the capillary 106 as well as the volumeassociated with the membrane tubing. As described previously, the lumenof the capillary 106 can include a separation matrix. Under appliedvoltage, if the separation matrix includes no spacers, which areelectrolyte solution added to a capillary to electrophoretically blockspecific segments of the capillary from being used to focus analytes(e.g., Iminodiacetic acid (IDA) and/or Arginine), the length of the pHgradient can extend over the entire length of the capillary and thelength of the portion of tubing that includes the volume associated withthe membrane tubing. The inner diameter and length of the membranetubing can thus affect the distribution of the pH gradient,subsequently, the quality of separation as well as the quality ofmobilization. Therefore, the volume associated with the membrane tubingcan be an important factor in the quality of separation and/ormobilization. Additionally, the inner diameter of the porous membranetubing can be selected to match the outer diameter of the capillary 106such that the junction between the membrane tubing and the capillary 106is sealed and does not become a defect area that may encouragepotentially detrimental effects (e.g., bubble formation).

In some embodiments, the capillary 106 can include one or more valveslocated at any suitable position along the length of the capillary 106and operational to open and/or close fluid flow through the capillary106 at any desired time. For example, the capillary 106 can include avalve located at or near a distal end such that the valve can be openedduring loading the capillary 106 with a sample and the valve can beclosed following loading and while separating the analytes (e.g., viaisoelectric focusing) to reduce or minimize hydrodynamic flow (e.g.,flow caused by gravity due to the vertical orientation of the capillary106) during separation. In some embodiments, the capillary 106 and/orthe cartridge 104 can be configured to include one or more adaptationsto permit an application of electric potential across the lumen of thevertically oriented capillary 106 via the first or top running bufferreservoir and the second or bottom running buffer reservoir, asdescribed in further detail herein. For example, in some embodiments, asdescribed previously, the capillary 106 and/or the cartridge 104 caninclude one or more electrical contacts that can be used to connect to asource of electrical power. For example, the capillary 106 and/or thecartridge 104 can include one or more electrical contacts disposed at ornear the proximal and and/or the distal end of the capillary 106.

The sample plate assembly 107 of the system 100 can be any suitableshape, size, or configuration and can be arranged to receive, house,and/or store at least a portion of a sample plate or a reagent tray (notshown in FIG. 1 ). For example, the reagent tray or sample plate canhold and/or otherwise define a set of vials, wells, well plates,microwell plates, troughs, and/or the like (any of which may begenerically described as a “well” or “microwell”). The wells and/ormicrowells can be any suitable size and can be disposed along and/orotherwise defined by a surface of the reagent tray in any suitablearrangement. Although specific examples of reagent trays are describedherein, the sample plate assembly 107 can be configured to receiveand/or include any suitable reagent tray of similar size and/or shapethat can define any number and/or any arrangement of wells and/ormicrowells. The wells and/or microwells included in or defined by thereagent tray can contain and/or receive any suitable volume of asolution, fluid, gel, lysate, buffer, sample, analyte, ampholyte, agent,reagent, protein, matrix, and/or the like. In some embodiments, thewells and/or microwells can receive a vial or the like containing avolume of any suitable fluid. In some embodiments, the sample plateassembly 107 and/or a portion of the sample plate assembly 107 iselectrically conductive and electrically coupled to the electricaland/or electronic assembly 108 included in the system 100. In suchembodiments, the sample plate and/or a portion of the sample plate canalso be electrically conductive and can facilitate an electricalconnection to a fluid disposed within the sample plate through thecoupling of the sample plate to the sample plate assembly 107.

At least a portion of the sample plate assembly 107 is movably disposedwithin and/or movably coupled to the housing 101. In some embodiments,the sample plate assembly 107 can be movably disposed in the housing 101such that the system 100 can automatically or semi-automaticallymanipulate a sample plate with respect to the cartridge retainer 103.The sample plate assembly 107 can be configured to move relative to thecartridge retainer 103 to place the capillary 106 of the cartridge 104in fluid communication with a reagent or sample or buffer volume in oneor more vials or wells in a sample plate manipulated by the sample plateassembly 107. In some embodiments, the system 100 can be configured suchthat the sample plate assembly 107 can be moved relative to thecartridge retainer 103 such that the distal end of the capillary 106 canbe sequentially disposed in a set of vials or wells that include asample, a running buffer, an elution reagent, a chemical mobilizer, andor the like.

For example, the sample plate assembly 107 can be movably coupled to oneor more tracks, racks, lead screws, slides, pistons, and/or the likethat can be operable to move the sample plate assembly 107 relative tothe housing 101. The sample plate assembly 107 (or at least a samplevial or at least a reagent tray included therein) can be moved in adirection closer to or further from the cartridge retainer 103, asindicated by the arrow AA in FIG. 1 . In other words, the sample plateassembly 107 can be moved in a direction parallel to an axis defined bythe capillary 106 of the cartridge 104 when the cartridge 104 isretained by the cartridge retainer 103. In addition, the sample plateassembly 107 can be moved in one or more directions along a plane normalto the cartridge retainer 103, as indicated by the arrow BB. That is tosay, the sample plate assembly 107 (or the sample plate included thereinor at least a vial or well from the sample plate) can be moved along aplane normal to the axis defined by the capillary 106 of the cartridge104 when the cartridge 104 is retained by the cartridge retainer 103.Said another way, the sample plate assembly 107 (or the sample plateincluded therein or at least a vial from the sample plate) can be movedwithin the housing 101 in the X-direction (e.g., left or right), theY-direction (e.g., up or down), and the Z-direction (e.g., front orback) relative to the cartridge retainer 103.

In this manner, the sample plate assembly 107 is configured to move atleast the sample plate or at least a vial or well from the sample plate(not shown in FIG. 1 ) relative to the cartridge 104 retained by thecartridge retainer 103 to dispose at least a distal end portion of thecapillary 106 of the cartridge 104 in the wells, microwells, vials,and/or the like of the sample plate. Moreover, the sample plate assembly107 can move at least the vial well or sample plate through any suitablenumber of positions relative to the cartridge 104 and/or cartridgeretainer 103 to place the capillary 106 in any of the wells, microwells,and/or vials included in the sample plate, or any suitable combinationthereof. The sample plate assembly 107 can be moved to drawsolutions/sample into the capillary 106 and/or to elute fractions fromthe capillary 106 and into collection wells, using any suitable methodincluding pressure-based injection, pressure-based elution, chemicalinjection, chemical mobilization, electrochemical injection,electrochemical mobilization, etc.

For example, with the capillary 106 in fluid communication with thevacuum source (as described above), a negative pressure can be producedwithin the capillary 106 that is operable to draw a volume of fluid,such as those described above, from any suitable well or wells of thereagent tray and into the capillary 106. In some instances, an electricfield can be applied across the lumen of the capillary 106, which canapply an electrokinetic force is operable to draw charged moieties intothe capillary 106 via electrokinetic injection, from any suitable wellor wells of the reagent tray and into the capillary 106. In someembodiments, a well, microwell, vial, etc., can be fluidically coupledto a positive pressure source via a pressure conduit (not shown in FIG.1 ) inserted into the well, microwell, vial etc. with the capillary 106.The sample plate assembly 107, the capillary 106, and/or a portion ofthe cartridge 104 can be operable to seal the well, microwell, vial,etc. against the cartridge 104 such that a positive pressure can urgefluid from the well, microwell, vial etc. into the capillary 106.

In some embodiments, the system 100 can be configured to manipulate thesample plate assembly 107 such that a portion of the distal end of thecapillary 106 can be disposed in a sample reservoir in a sample plate ata first time and the suction or electrokinetic force is applied to drawthe sample, and then the capillary 106 can be disposed in a in a secondrunning buffer reservoir in a sample well plate. In some instances, thesample plate assembly 107 can be configured to be manipulated such thatthe proximal end of the capillary distal end capillary 106 can beconfigured to be disposed in sample reservoir in a sample well plateand/or a buffer reservoir in a sample well plate. Suction appliedthrough a sheath interface of the capillary 106 or a tubing coupled tothe proximal end of the capillary 106 via the vacuum source can drawsample/buffer from such reservoirs and bring the sample/buffer into thelumen of the capillary 106. Then the system 100 can be manipulated toperform a separation of analytes in the sample (e.g., via isoelectricfocusing by applying a voltage across the proximal and distal ends ofthe capillary while top and bottom running buffers induce a pH gradientacross the capillary). The separated analytes can be analyzed todetermine a degree of separation that can be measured between eachseparated analyte. The analysis can be conducted using the probe system102 and/or electronic system 108.

The separated analytes can be mobilized to be eluted using any suitablemechanism or driving force. For example, the separated analytes can bemobilized to migrate towards the distal end of the capillary 106 at aspecified rate using an applied positive pressure at the proximal end ofthe capillary 106. Based on the measured separation between analytes anexpected rate or duration of elution of each analyte can be calculatedand the sample plate assembly 107 can be manipulated to introduceisolated collection vials or collection wells in the sample plate toreceive each eluted analyte or fraction as a product of fractionation.In some instances, the analytes separated in the capillary 106 can besimultaneously or near-simultaneously detected and/or visualized duringthe elution phase to monitor a degree of separation and/or a relativelocation of each analyte (e.g., peak of each distribution correspondingto each analyte)

As another example, the separated analytes can be induced to migratetowards the distal end of the capillary 106 by manipulating the sampleplate assembly 107 such that a set of collection wells or a collectionvials having chemical mobilizers are sequentially introduced for thedistal end to be disposed in. The set of collection well with chemicalmobilizers may be configured such that each collection well includes achemical mobilizer (e.g., the same or different chemical mobilizers)configured to disrupt the pH gradient formed during separation. Thechemical mobilizers can be carefully selected to induce chemicalmobilization to draw out and elute the most distal separated analyte oranalytes. The chemical mobilizers can be used to carefully draw thedesired portion of the separated sample while not substantially drawingthe next subsequent separated analyte that is undesired to be mixed withthe collected fraction of analyte or analytes. For example, eachcollection well can contain a buffer configured to incrementallydecrease the pH gradient across the capillary such that analyte(s)migrate towards the sample plate and/or into the collection well(s).

In some instances, one or more of the separated analytes can beelectrochemically mobilized and/or induced to migrate towards the distalend of the capillary 106 to be eluted. The elution can be monitored,and/or controlled as desired. For example, the capillary 106 can bevisualized at a viewing window and a rate of mobilization and/or a rateof elution can be controlled by modifying an elution rate, an elutionvolume, an elution duration, etc. Any suitable driving force can be usedincluding pressure-based elution, chemical mobilization, electrochemicalmobilization, etc. In some instances, the distal end of the capillary106 can be disposed in a collection well with a second running bufferdifferent from the first running buffer in a running buffer reservoir105 (e.g., an acid in an acid tank) in which the proximal end of thecapillary 106 is disposed and applying an electric field.

A sequence of collection wells can be included in a sample plate witheach collection well including a chemical mobilizer such that the distalportion of the capillary 106 can be sequentially disposed in eachsuccessive collection well. To elute fractions using chemicalmobilization, when the distal end of the capillary 106 is disposed ineach well an electric field can be applied such that separated analytesfrom the distal end to the proximal end of the capillary 106 can besequentially eluted into each successive collection well. In someinstances, optionally the speed of elution can be varied (e.g.,increased or decreased) during the elution phase, based on selection ofchemical mobilizers, use of additional hydrodynamic mobilization, etc.,to further control elution and/or fraction collection (e.g., speed up,slow down, and/or cease elution, etc.). Chemical mobilizers can beselected to completely elute analytes separated along a pH gradient, forexample, by sequentially disposing a distal end of the capillary incollection wells containing a chemical mobilizer until all the analytesare eluted in fractions. The migration of a target fraction can bemonitored as it migrates towards a target collection well. A samplecollection plate can move to place a distal end of the capillary 106into a different collection well containing a chemical mobilizer at eachinstance when a separate fraction is to be collected. Voltage can beapplied when the distal end of the capillary is disposed in thecollection well such that the target fraction can be migrated towardsthe distal end of the capillary 106 and eluted out into the collectionwell. This process can be completed to collect any suitable number offractions.

In some instances, optionally, a positive or negative pressure can beapplied during elution to either further induce migration and elution orto counteract migration (e.g., hydrodynamic migration or migration dueto effects of gravity) at various stages of the elution phase orfraction collection phase. In some instances, a simple pressure-basedmethod can be used by itself to monitor and control elution and fractioncollection. For example, a small negative pressure or vacuum can beapplied (at the proximal end of the capillary 106) at all times exceptwhen the distal end of the capillary 106 is disposed in a collectionwell. The contents of the capillary 106 can be monitored when the distalend if disposed in a collection well and a small positive pressure canbe applied while monitoring mobilization of the contents of thecapillary 106. The positive pressure can be ceased and/or switched withthe small negative pressure, as soon as the desired analyte or analytesis collected in the collection well to prevent contamination by otherunwanted contents of the capillary 106. The amount of positive and/ornegative pressure, the duration and time of application, and the likecan be chosen depending on parameters like dimensions of the capillary,material properties of the buffers, sample, etc. used, a degree ofseparation of analytes, a location of each separated analyte, anidentity of a desired analyte or analytes to be collected as a fraction,and so on.

Each running buffer or chemical mobilizer in the collection wells,pressure, and/or an electric field to be applied when the distal end ofthe capillary 106 is disposed in each running buffer in the collectionwells can be monitored while eluting to carefully control a rate ofelution or volume of elution or a time of elution. The properties of thebuffer or chemical mobilizer, the amount of pressure and time ofapplication, and/or the magnitude and time of application of the voltageor electric field can be carefully selected to draw out only the mostdistal separated analyte or analytes defined by an desired point ofseparation located at a specified position along the length of thecapillary 106. The running buffers and/or the applied electric fieldscan be used carefully draw the desired fraction of the separatedanalytes in the sample while not substantially drawing or disturbing thesuccessive separated fractions of analyte or analytes that are not to bemixed with the collected fraction of analyte or analytes.

The sample plate assembly 107 and or the capillary 106 can bemanipulated such that the distal end of the capillary 106 is disposed ina sequence of collection well in a sequential manner while elutingseparated analytes in successive collection well of the sample plate. Insome embodiments, the system 100 can be configured such that the sampleplate assembly 107 can be automatically manipulated at a specified rateof introducing each successive collection well the rate being determinedby a real-time analysis of the degree of separation of analytes. Asanother example, the sample plate assembly 107 can be manipulated to asequence of collection well wherein the distal end of the capillary 106can be disposed. A sequence of voltages or electric fields of aspecified magnitude can be applied such that at each collection well themost distal set of analytes (e.g., analytes with the highest or lowestremaining pI, as is applicable in an implementation) desired to beisolated are collected upon applying the electric field. In someembodiments, as described previously, the sample plate assembly 107 canbe automatically manipulated to introduce and/or position eachsuccessive collection well at a specified rate of movement to optimallyisolate each separated analyte or set of analytes, the rate beingdetermined by a real-time analysis of the degree of separation ofanalytes.

In some instances, a user can load a capillary cartridge 104 into thesystem 100 and can initiate and/or otherwise provide instructions to thesystem 100 to cause the system 100 to at least semi-automaticallyseparate analytes within the sample by isoelectric point. In someinstances, the system 100 draws a sample (e.g., including any suitableagent, reagent, protein, analyte, buffer, lysate, etc.) into acapillary, separates and/or focuses analytes in the sample within thecapillary, and detects the presence or the absence of a target analyteand/or detects the location of analytes within the sample (e.g.,analytes that have migrated to different positions along the capillaryassociated with their isoelectric points). The system 100 can thenselectively elute at least some constituents of the sample within thecapillary (e.g., one or more separated analytes) in a serial manner suchthat the separated analytes can be collected in collection wells and beused for further processing.

In use, for example, the system 100 can be set, programed, and/orotherwise placed in a configuration to perform fractionation of asample, which can include, for example, preparing samples and/orreagents as well as preparing the cartridge 104. A user can then insertthe cartridge 104 into the cartridge retainer 103 in a single,predetermined orientation, as described above. The cartridge retainer103, in turn, at least temporarily couples to the cartridge 104 toretain the cartridge 104 in a substantially fixed (e.g., verticallyoriented) position. The cartridge retainer 103 can couple to thecartridge 104 such that the proximal end of the capillary 106 isdisposed in a first running buffer (e.g., acid of a desired acidic pH)held in a running buffer reservoir 105. The proximal portion of thecontents of the lumen of the capillary 106 can be electrically andionically coupled to the first running buffer via electrical contactsand/or fluidic connection. The sample plate assembly 107 is movedrelative to the capillary 106 such that a distal end portion of thecapillary 106 is disposed in a first vial or well containing a samplesolution. A suitable driving force, for example, a vacuum source, isused to draw and load the sample into the capillaryl06. The drawing andloading the sample can be accompanied by suitably operating one or morevalues that may be associated with loading the sample. For example, avalve in the distal end of the capillary 106 can be opened to allow thesample to be loaded while a valve in the proximal end can be closed toreduce movement of first running buffer into the capillary 106. Thesample plate assembly may then be operated to remove the first vial orwell and introduce a second well or vial that can include a secondrunning buffer in which the distal end of the capillary 106 is disposed.The distal portion of the contents of the lumen of the capillary 106 canbe electrically and ionically coupled to the second running buffer viaelectrical contacts and/or fluidic connection. The electronic system 108can provide instructions to apply and/or apply an electric field betweenthe first running buffer and the second running buffer via the contentsof the lumen of the capillary 106. The applied electric field can induceseparation of one or more analytes in the sample via isoelectricfocusing. The analytes can be charge variants each associated with adifferent charge such that each separated analyte migrates and localizesat a point that corresponds to its isoelectric point (of neutralizedcharges) along the length of the capillary 106. The first running bufferand the second running buffer can be selected such that they define a pHrange that encompasses the isoelectric points (pI) of the analytes orfractions that are desired to be separated and fractionated from thesample.

Following separation, the probe system 102 can be used to analyze theseparated analytes using any suitable mechanism (e.g., fluorescenceemission analysis, optical density analysis, etc.) The arrangement ofthe cartridge retainer 103 and the probe system 102 within the housing101 is such that predetermined portions of the probe system 102 (e.g.,an emitter and a detector) are aligned with predetermined portions ofthe cartridge retainer 103 (e.g., one or more openings or viewingwindows or the like). Thus, by aligning the probe system 102 with thecartridge retainer 103 and with the cartridge retainer 103 retaining thecartridge 104 in a predetermined, fixed position, the emitter and thedetector can be aligned with, for example, a portion of the capillary106 of the cartridge 104. Therefore, energy and/or light emitted by thefirst emitter can be directed to a predetermined portion or length ofthe capillary 106. The detector can capture the signal emitted by theseparated analytes and analyze the captured signal to determine a degreeof separation, location of separation (e.g., relative location of peakquantity or a defined portion of a quantity) of a separated analytealong the length of the capillaryl06, and/or an identity of theseparated analyte(s).

In some instances, a full-column detection portion of a detector candetect “full-column” images or signals and/or otherwise can perform“full-column” detection of the sample within the capillary 106.Similarly stated, the detector can include an imaging device included inthe probe system 102 can be operable to capture more than a single pointalong the capillary 106. For example, the imaging device can be operableto capture and/or detect a sufficient length of the capillary 106 tovisualize separation and/or focusing of analytes during the separationand/or mobilization process (e.g., a length of about 1 cm, about 3 cm,about 5 cm, about 10 cm, about 20 cm, about 50 cm, or any other suitablelength of the capillary 106). In addition, or alternatively, the imagingdevice can be operable to capture and/or detect native fluorescence, andabsorbance of analytes within the capillary 106. For example, the probesystem 102 can include a filter wheel associated with the detectorand/or emitter such that the filter wheel can be rotated to change theoptical signal presented to the sample and/or received from the samplewhile analytes are being separated, focused, and/or mobilized within thecapillary 106. Thus, during a single run, a sample can be characterizedfor native fluorescence, absorbance, and/or any other suitable opticalcharacteristic along the full-column while the analytes separate,focused, and/or are mobilized to be eluted in fractions.

The sample plate 107 can be operated, in conjunction with the analysisby the probe system 102 to dispose the distal end of the capillary 106in successive collection wells including running buffers and/or chemicalmobilizers that can be used to draw and elute a fraction of theseparated analytes by applying a suitable driving force such as positivepressure by the vacuum source, or an applied electric field of aspecified magnitude, and/or a chemical mobilizer with a desired pH. Thesample plate 107 can be moved at a specified rate based on a real-timepeak analysis (analysis of relative location of peaks of separatedanalytes), or an analysis of relative distribution of quantities ofseparated fractions of the analytes. The fractions once collected canthen be used for any further downstream processing as desired.

Cartridge Including a Capillary with a Single Membrane

In some embodiments, a cartridge can be configured to include a singleporous membrane. In some implementations, the cartridge can becompatible with systems configured for separation of analytes viaisoelectric focusing of samples with the analytes (e.g., proteins) andother collection of charge variants (e.g., Maurice™ systems byProteinSimple®) with a capillary fixed inside the cartridge. One end ofthe capillary, for example the proximal end, can include a small portionof a porous membrane tubing. The membrane tubing can be configured to beconnected to a vacuum source, which can help draw a sample into thecapillary through the distal open end of the capillary tube. The porousmembrane can allow exchange of ions between the first running buffer inthe running buffer reservoir (e.g., acid in an acid tank, which can beintegrated or included in the cartridge), and the sample inside thecapillary during the separation phase, via isoelectric focusing andduring a chemical mobilization phase. A small portion of the distal openend of the capillary can protrude out of the cartridge cover. Theprotruding portion can be housed within a metal orifice tip, asdescribed in further detail with reference to some embodiments disclosedbelow. Such a cartridge can be configured to implement the followingsteps. (1) A sample can be loaded into the capillary by application ofvacuum or pressure when the metal orifice tip and the open distal end ofthe capillary is disposed or dipped in a well or vial containingsamples. (2) A sample drawn inside the capillary lumen can beisoelectrically focused when a voltage is applied between the firstrunning buffer in the running buffer reservoir (e.g., acid tank) andmetal tip while the metal tip is disposed in a well or vial containing asecond running buffer (e.g., base solution). (3) Separated chargevariants can be mobilized out of the capillary into a collection wellwhen a voltage is applied between the first running buffer reservoir(e.g., acid tank) and the metal tip while the metal tip is disposed in awell or vial containing a chemical mobilizer. The porous membrane can bedesigned and/or selected to efficiently balance the ion-exchangesbetween the first and second running buffers and/or the first runningbuffer and the chemical mobilizer. For example, the porous membrane canefficiently balance the ion-exchanges between the acid in the acid tankand the base solution in the vial. In some embodiments, the porousmembrane can be selected to reduce or minimize any potentialhydrodynamic flow during the isoelectric focusing stage (e.g., flowcaused by gravity due to a vertical orientation of the capillary usedfor separation). In some embodiments, the membrane can be designed toenable effective mobilization speed and minimize the disruption of thepH gradient during the mobilization stage.

Fractionation System Using a Capillary Including a Single Segment

In some embodiments, a fractionation system can use a capillarycartridge with a capillary with a single inner diameter as describedpreviously with reference to the system 100. FIG. 2 is a schematicrepresentation of a capillary cartridge 204, according to an embodiment.The cartridge 204 can be substantially similar in structure and/orfunction to the cartridge 104 described above and can be used by afractionation system substantially similar to the system 100 describedabove.

The cartridge 204 includes a running buffer reservoir 205 and acapillary 206 disposed in the running buffer reservoir 205 such that thelumen of the capillary 206 at the proximal portion of the capillary 206is configured to be in fluidic and ionic connection with the runningbuffer in the running buffer reservoir 205.

The running buffer reservoir 205 can be substantially similar instructure and/or function to the running buffer reservoir 105 describedpreviously with reference to the system 100. The running bufferreservoir 205 can include a first running buffer of a first pH. Forexample, the running buffer reservoir 205 can include an acid of aspecified acidic pH. The running buffer reservoir 205 can have anysuitable volume. For example, it can have a volume of 500 μl to 4000 μl.

The capillary 206 can be substantially similar in structure and/orfunction to the capillary 106 described previously with reference to thesystem 100. The capillary 206 can be of any suitable length. Forexample, in some embodiments, the capillary 206 can have a length of 5mm-1 m. In some embodiments, the capillary 206 can have a length of50-120 mm. In some other embodiments, as described in further detailbelow, the capillary 206 can be longer at between 20 to 30 cm length. Insome other embodiments, the capillary 206 can be even longer at ˜1 mlength. The capillary 206 can have any suitable inner diameter. In someembodiments, the capillary 206 can have an inner diameter of 320-530 μm.In some embodiments, the capillary 206 can be used to achieve highresolution of separation of larger samples and have an inner diameter of200 to 500 μm.

The capillary 206 includes a porous membrane 213 disposed at theproximal end of the capillary 206 and configured to serve as ahydrodynamic barrier between the running buffer in the running bufferreservoir and the lumen of the capillary 206 at or near the proximal endof the capillary 206. In some embodiments, the cartridge 204 can includea tubing 211 (e.g., an additional length or segment of connectingcapillary, a length of deformable polymer tubing (e.g., Tygon tubing),or a combination of such elements) that can be used to couple the porousmembrane 213 and the distal end of the capillary 206 to an externalvacuum source or pressure source. The external vacuum source or pressuresource can be used to draw a sample into the capillary 206 as describedpreviously with reference to the capillary 106 in the system 100 shownin FIG. 1 . In some implementations, the tubing 211 can also be used tocouple the lumen of the capillary 206 to a vacuum source and a wastereservoir (not shown in FIG.2), for example the tubing 211 can beconfigured to be in fluidic connection to the vacuum source and a wastereservoir, such that contents of the capillary 206 and/or the porousmembrane 213 can be drawn into the waste reservoir by providing suctionusing the vacuum source. The tubing 211 can include or run through oneor more values (e.g., pinch valves) to control and/or route flow offluid via the tubing 211 (e.g., into the waste reservoir).

In some embodiments, the porous membrane 213 can be disposed at aproximal end of the capillary 206 and the tubing 211 can be disposed ata proximal end of the porous membrane 213 such that the capillary 206,the porous membrane 213, and the tubing 211 can form a continuous lumen.In some embodiments, the porous membrane 213 can be disposed at aproximal end of a segment of the capillary 206 and there can be anadditional segment of the capillary 206 (e.g., a segment of a connectingcapillary) coupled to the porous membrane 213 which can in turn becoupled to the tubing 211. Said in another way, the porous membrane 213can be disposed between two segments of the capillary 206, a segment forseparation and a segment of connecting capillary, such that the moreproximal segment of connecting capillary can be coupled to the tubing211 instead of the porous membrane 213 being coupled to the tubing 211directly. The additional segment of connecting capillary can permit morefacile connection between the porous membrane 213 (which can be a porousmembrane tubing) and the tubing 211. This configuration of using aconnecting capillary to couple the porous membrane tubing 213 to thetubing 211 can take advantage of the fact that the porous membranetubing is sized or selected such that it can seal and/or be glued to thecapillary 206 (e.g., the inner diameter of the porous membrane 213 canbe selected to form a tight seal with the outer diameter of thecapillary 206 including the segment of connecting capillary and thesegment of separation capillary). The tubing 211 (e.g., a soft siliconetubing) can be easily selected and used to push or clasp over thesegment of connecting capillary and form a tight seal. The tubing 211can be coupled to the vacuum source and/or the waste reservoir and aflow can be routed or controlled (e.g., using values) as describedherein.

Fractionation System Using a Capillary Including a Single Segment ofLarger Inner Diameter

In some implementations, to maximize the sample loading capacity and atthe same time maintain the advantage of real time monitoring of theisoelectric focusing progress (e.g., while using a system similar to theMaurice™ icIEF system), a capillary 206 with a large inner diameter(320-530 μm) with a given length (60-120 mm) can be used for theabove-described single membrane cartridge. In some implementations, itcan may be desirable to select the parameters of the capillary 206(e.g., inner diameter, outer diameter, length, etc.) in consideration ofand/or to match other parameters of the system, including a size of therunning buffer reservoir 205 (e.g., acid tank) which can be from 500 ulto 4000 ul, a membrane with an MWCO from 10 kDa to 500 kDa, a volumeassociated with the membrane tubing 213 which can be 300 um ID to 700 umID (with a length of membrane tubing between 0.5 mm to 2 mm). In someimplementations, the capillary 206 can have an inner diameter and/orouter diameter such that the efficiency of isoelectric focusing can bemaximized and/or the chemical mobilization current and the mobilizationspeed can meet a desired threshold value.

The porous membrane 213 can have a pore size defining a selectivity(i.e., MWCO) in allowing substances to flow or pass through. The MWCO ofa porous membrane 213 can be any suitable size between 10 kDa to 500 kDaselected based on the implementation. In some instances, the porousmembrane 213 can be selected based on or in conjunction with a selectionof a volume associated with the running buffer reservoir 205. In someinstances, the porous membrane 213 can be selected based on a deadvolume associated with the tubing 211 included in the porous membrane213 when the tubing 211 is coupled to the capillary 206. The dead volumecan be a volume of solution that remains in the tubing 211 included inthe porous membrane 213 after an operation (e.g., separation viaisoelectric focusing, or mobilization and elution of fractions, etc.).In some instances, the porous membrane 213 can be selected based on anouter diameter and/or an inner diameter of the capillary 206. Forexample, the porous membrane 213, the running buffer reservoir 205,and/or the capillary 206 can be selected to match or complement eachother for a specified implementation. As an example, a capillary with aninner diameter 300 um inner diameter to 700 um inner diameter can bematched with a porous membrane with a tubing 211 that has a lengthbetween 0.5 mm to 2 mm associated with a specified dead volume. Such aselection of the porous membrane 213, capillary 206, running bufferreservoir 205, and/or with the outer diameter and inner diameter of thecapillary 206 can be implemented to achieve an increased or targetefficiency of separation of analytes using isoelectric focusing and/orto achieve a desired chemical mobilization current / mobilization speedthat is optimal for eluting desired fractions from the sample.

The capillary 206 includes a metal tip 212 disposed at the distal end ofthe capillary 206. The metal tip 212 includes an orifice that is alignedwith the lumen of the capillary 206 and is configured to allow fluidicconnection between the lumen of the capillary 206 at the distal end withthe contents of a vial or well that the distal end of the capillary 206may be disposed in as described previously.

During separation the analytes in the sample held in the lumen of thecapillary 206 can be isolated or separated using isoelectric focusing bysubjecting the sample to a first electrical field. The first electricfield can be applied between the running buffer reservoir 205 holding afirst running buffer (e.g., an acid) ionically coupled to the proximalend of the lumen of the capillary 206 and a second running buffer in asample well or vial and ionically coupled to the distal end of the lumenof the capillary 206 via the metal tip 212 disposed in the secondrunning buffer (e.g., a base). On application of the electric fieldcurrent flows through the sample in the capillary 206 causing hydroniumions in the first running buffer (i.e. acid) held in the running bufferreservoir 205 start to migrate through the porous membrane 213 into thecapillary 206 and towards the cathode at the distal end of the capillary206, i.e. the portion of the capillary 206 comprising the metal tip 212disposed in the second running buffer (the base), and hydroxide ionsmove towards the anode at the proximal end of the capillary 206, i.e.the portion of the capillary 206 disposed in the first running buffer(the acid) to establish a pH gradient. As the pH gradient isestablished, ampholytic analytes in the capillary will experience aforce urging them to migrate towards a pH corresponding to their pI.Thus, the analytes with the lowest pIs will travel farthest towards theanode before they are neutralized by acquiring a zero-net charge. Whenanalytes/ampholytes get neutralized in charge their movement ceases andthey get focused at the point at which they are neutralized. Analyteswith higher pIs neutralize nearer to the distal end of the capillary 206(i.e., travels lesser than more acidic analytes/ampholytes).

Once separated, during chemical mobilization, the migrated peaks ofanalytes in the capillary 206 can be subjected to an influence of anapplied electric field to elute fractions of the analytes for furtherprocessing as described previously. The electric field used duringchemical mobilization can be applied between the running bufferreservoir 205 holding the first running buffer and the metal tip 212which may be disposed inside a collection well or vial containing achemical mobilizer.

A viewing window can be situated at any suitable location along thelength of the capillary 206 and be of any suitable size (length and/orwidth) such that a probe system can be used to detect the presenceand/or movement of separated analytes in the sample.

During the chemical mobilization phase, under the influence of voltageapplied between the first running buffer for example, the acid in theacid tank, and the metal tip 212 which is disposed inside a collectionwell containing the chemical mobilizer, the negative ions of themobilizer in the well can move into the lumen of the capillary 206 andtowards the membrane side of the capillary 206 toward the acid tank 205,disrupting the pH gradient formed during the separation stage, causinganalytes to migrate toward the cathode as they obtain a positive charge.Thus, in some instances, in practice, mobilization can cause a pHchange, particularly at the distal end of the capillary 206, where ionsfrom the chemical mobilizer move into the capillary 206. Thus, a changeof the pH gradient during mobilization can be unavoidable.

The addition of the chemical mobilizer can cause the analytes with thehighest pIs to be eluted from the capillary and collected in acollection well. The rate of change of pH gradient, if too fast, can bedetrimental on the resolution of collected fractions. The rate of changecan depend on the amount of ions moving into the capillary 206, themobility of the ions, and/or the buffering capacity of the carrierampholytes (see for example, Rodriguez-Diaz, R., Wehr, T., Zhu, M.,Levi, V., Handbook of Capillary Electrophoresis (2nd Edition) 1997,101-138, the entire contents of which are hereby incorporated byreference). The inventors of the instant application observed that thespeed of peak mobilization can be controlled by the concentration of thechemical mobilizer as well as the voltage. For example, higherconcentration and/or greater voltage lead to higher speed peakmobilization. In practice, the concentration of the mobilizer and/or themagnitude of mobilization voltage can be selected to achieve a desiredspeed of mobilization that can fall between 1 mm/min and 2 mm/min. Withknowledge of the position (along the length of the capillary 206 and/orthe pH gradient) of a peak of an analyte, the width of the peak, and thespeed of peak mobilization, one can selectively collect a desiredanalyte associated with a peak into a single fraction or multiplefractions of desired purity by adjusting the time window of fractioncollection.

In some implementations, a single mobilizer can be used whose pH islower than the pI of all the analytes to be separated and eluted. Thechemical mobilizer can titrate the pH gradient inside the capillary 206gradually from the higher pI at the distal end toward lower pI at theproximal end. During such titration process, the peak with higher pI canbecome positively charged first and therefore be eluted out of thecapillary 206 into the collection well first. In some implementations,the electric field and/or pH of the mobilizer can be kept constant. Insome embodiments, the voltage and/or the pH of the chemical mobilizercan be changed at each elution of a fraction (e.g., elution associatedwith each collection well) as desired.

The steps followed during mobilization of fractions can be such that thefocused or separated peaks of analytes can maintain their resolution ofseparation and/or relative separated position along the pH gradientduring the migration and/or elution of fractions. In some instances,mobilization and/or elution may cause a change in the pH gradient, forexample at the distal end of the capillary 206, where ions from achemical mobilizer may move into the lumen of the capillary 206. In somesuch instances, a fast rate of change of pH gradient (e.g., above aspecified threshold rate of change), may be detrimental to theresolution of separated analytes and/or collected fractions. The rate ofchange of the pH gradient can depend on the number of ions migratingfrom a chemical mobilizer and into the lumen of the capillary 206, amobility of the ions, and a buffering capacity of a carrier ampholytespresent in the sample in the capillary 206.

In some embodiments, a rate at which a peak quantity associated with aseparated or isolated quantity of an analyte can be mobilized ormigrated towards a collection well (also referred to as peakmobilization) can be modulated and/or controlled by a concentration ofions in the chemical mobilizer in the collection well used and/or thevoltage applied to promote mobilization. For example, a higher negativeion concentration in the chemical mobilizer can lead to higher speed ofpeak mobilization. The ion concentration of chemical mobilizer can bebetween 1 mM to 200 mM. As another example, higher magnitude appliedvoltage (creating a higher electric field) can lead to higher speed ofpeak mobilization. The voltage can be between 500V to 2000V. Thenegative ion concentration of the chemical mobilizer and the magnitudeof mobilization voltage can thus be selected to make the speed ofmobilization of one or more analytes fall between 0.5 mm/min and 2mm/min. The collection times (time to collect desired target fractionsfrom a sample) can be between 30 s to 300 s. In some instances, afractionation system (e.g., systems 100,200) can be used to determine aposition of a peak associated with a quantity of a separated analyte, awidth associated with the peak quantity of the separated analyte, and/orthe speed of peak mobilization associated with one or more analytes.With this information, the fractionation system can be used toselectively elute and collect a desired peak into a single or multiplepredefined fractions by adjusting the time window of fraction collection(e.g., coordinated with a rate of movement of a sample plate assembly asdescribed previously).

Fractionation System Using a Capillary Including a Single Segment ofLonger Length

In some embodiments, using a capillary of smaller inner diameter andlonger length can permit elution of purer fractions of individual peaksof analytes that have been separated. In some embodiments, a cartridgecan include a capillary 206 with an inner diameter of 200-500 um and atotal length of the separation capillary from 20-30 cm. In someembodiments, the initial sample focusing profile may be outside theviewing window of the detector so that a portion or all of the samplepeaks may not be visible when the separation begins, depending on the pIand/or pI range of the analytes in the sample. The peaks of the desiredanalytes may, however, appear in the viewing window during samplemobilization and elution and the full sample focusing profile can bereconstructed later through image processing. In other words, an imagingsystem can act as a composite full-field and point detector when usedwith a cartridge with a long capillary. To speed up the samplemobilization and elution for such a cartridge, in some implementations,the system can be configured to introduce hydrodynamic mobilization inaddition to chemical mobilization. To enable hydrodynamic mobilization,the cartridge 204 can include a large pore size membrane (MWCO fromabout 100 to 1000 kDa) between a proximal end of the capillary and therunning buffer reservoir at or above the capillary (e.g., a top runningbuffer reservoir) and/or a particular location and/or orientation of therunning buffer reservoir to encourage hydrodynamic mobilization asdescribed herein. For example, in some embodiments, the porous membranecan have suitably large pore size and the running buffer reservoir canbe located vertically above the proximal end of the capillary 206 suchthat the first running buffer in the running buffer reservoir (e.g.,acid in the acid tank) can flow through the porous membrane and into thecapillary 206 due to gravity. To manage and control the flow rate of theacid, a source of low vacuum can be connected to the acid tank which canoppose the gravitational force on the fluid in the capillary 206. Duringthe initial sample focusing phase, the low vacuum can be increased sothat there is no or minimal flow in the capillary 206. After thefocusing is complete, the low vacuum can be reduced to allow acid flowinto the capillary to initiate the hydrodynamic mobilization. In someimplementations, the voltage can be applied concurrently withhydrodynamic mobilization to maintain separation resolution duringmobilization of analytes. When the distal end of the capillary isdisposed in a running buffer for separation (e.g., a base), then theapplied electric field can result in maintenance of the pH gradientduring the hydrodynamic mobilization. If the distal end of the capillaryis disposed in a chemical mobilizer for mobilization/elution, then therewill be a combination of hydrodynamic and chemical mobilization. Bymonitoring the sample peak migration in the viewing window, the systemcan be configured to calculate the hydrodynamic mobilization speed.Based on the speed of mobilization a time of arrival of individual peaksof interest in the sample at the distal end of the capillary end can bepredicted so that the sample elution for that fraction of interest canbe started. During sample elution, both hydrodynamic and chemicalmobilization can act in concert when the elution buffer is also achemical mobilizer.

Fractionation System Using a Capillary Including Multiple Segments

One of the performance parameters of a fractionation device is theresolution of the collected fractions achieved by the fractionationsystem. Resolution of collected fractions can be analyzed as follows.Consider two example analytes that when separated, for example usingisoelectric focusing, can form two peaks of separated quantities (A andB) with neighboring pI values, such that pI_(A)<pI_(B). The separatedanalytes can be eluted and collected in two consecutive collection wells(1 and 2). Separation resolution can be defined as the smallest pIdifference, ΔpI=pI_(B)−pI_(A), for which the percentage of peak A incollection well 1 and the percentage of peak B in collection well 2 areboth >85%.

Another important performance parameter of a fractionation system is thefraction recovery yield, which is the ratio of the collected amount ormass of a sample component to the amount or mass of the sample componentthat was loaded into the capillary. When using a fractionation systemconfigured to use a capillary cartridge that includes a capillary with afixed length, the amount of sample that can be loaded in a capillarywith the larger inner diameter can be greater than the amount of samplethat can be loaded in a capillary with a smaller inner diameter for thesame length due to a reduction in volume.

As described above, using a capillary with a relatively large innerdiameter allows a higher sample loading capacity compared to a capillarywith a smaller inner diameter. A capillary with a larger inner diameter,however, may have a lower separation resolution as well as lowerrecovery yield. A capillary with smaller inner diameter can supporthigher separation resolution but may be limited in sample loadingcapacity unless capillaries of much larger length are used. To achieveboth a higher sample loading capacity and higher separation resolutionat a given length, some embodiments of a fractionation system can beconfigured to use a capillary cartridge including a capillary with twoor more segments (i.e. a plurality of segments) each segment beingassociated with a specified length and a specified inner diameter.

FIG. 3 shows a schematic representation of a capillary cartridge 304that includes a capillary with multiple segments, according to anembodiment. The capillary cartridge 304 can be substantially similar instructure and/or function to the cartridge 104 and/or the cartridge 204described previously and can be used by a fractionation systemsubstantially similar to the systems 100, 200 described above. Thecartridge 304 includes a running buffer reservoir 305, a capillary 306,a porous membrane 313, a tubing 311, and a metal tip 312. Additionally,the capillary 306 includes three segments a first segment 306 a, asecond segment 306 b, and a third segment 306 c.

As described previously, the capillary 306 can include multiple (i.e.,at least two) segments that each have a specified length and innerdiameter. The capillary cartridge 304 including the capillary 306combines the advantage of high sample loading capacity of a capillarywith a segment 306 a having a larger inner diameter, and the advantageof better resolution and fraction recovery yield of a capillary with asegment 306 c having a smaller inner diameter, and includes a segment306 b that has a variable inner diameter that transitions from that ofthe segment 306 a to that of segment 306 c as illustrated in FIG. 3 .

The segment 306 a, or a first segment, of the capillary 306 is locatedtowards the proximal end of the capillary 306 near the running bufferreservoir 305. The proximal end of the segment 306 a is configured toinclude and/or be coupled to the porous membrane 313 and to be disposedin the running buffer reservoir 305 as described previously withreference to the capillary 106 and/or 206 in the systems 100 and/or 200herein. The porous membrane 313 can be coupled, affixed (or the like) toa tubing 311 (e.g., soft silicone tubing), a length or segment of aconnecting capillary followed by a length of tubing 311 (e.g., softsilicone tubing) which can then be coupled to an externalpressure/vacuum source, and/or a waste reservoir. The tubing 311 can becoupled to an external pressure source or vacuum source as describedpreviously with reference to the cartridge 104 and/or 204. The segment306 a can have a proximal end disposed in the running buffer reservoir305 such that the lumen of the capillary 306 at the proximal portion ofthe segment 306 a of the capillary 306 is configured to be in fluidicand ionic connection with the running buffer in the running bufferreservoir 305. The segment 306 c can have a distal end coupled to themetal tip 312 and configured to be disposed in a running buffer ofchemical mobilizer in a sample well or vial (e.g., operated by a sampleplate assembly) such that the lumen of the capillary 306 at the distalportion of the segment 306 c of the capillary 306 is configured to be influidic and ionic connection with the running buffer or chemicalmobilizer in the sample well/vial or collection well/vial.

The segment 306 a of the capillary 306 has an inner diameter larger thanthe segment 306 b of the capillary 306 that is at the distal end andnear the metal tip 312. In some embodiments, the inner diameter of thesegment 306 a can be any suitable value between 300 and 500 μm. Thelength of the segment 306 a can be any suitable value between 20 cm to60 cm.

The segment 306 c, or a third segment, of the capillary 306 is locatedtowards the distal end of the capillary 306 and is coupled to the metaltip 312 which can be dipped or disposed in a well or vial holding arunning buffer and/or chemical mobilizer, to induce separation ofanalytes or mobilization of separated analytes, as described previouslywith reference to the cartridge 104 and/or 204. The segment 306 c of thecapillary 306 can have an inner diameter that is smaller than thesegment 306 a of the capillary 306 that is at the proximal end anddisposed in the running buffer reservoir 305. In some embodiments, theinner diameter of the segment 306 c can be any suitable value between100 and 200 μm. The length of the segment 306 a can be any suitablevalue between 20 cm to 60 cm. The length of the segment 306 c can be anysuitable value between 1 cm to 20 cm.

The segment 306 b, or the second segment, of the capillary 306 islocated between the segment 306 a and the segment 306 c and serves as atransition segment with its inner diameter being variable. The innerdiameter of the segment 306 b can be configured to change from a valueclose to the inner diameter of the segment 306 a, at a portion proximalto the segment 306 a, to a value close to the inner diameter of thesegment 306 c, at a portion proximal to the segment 306 c. The length ofthe segment 306 b can be any value determined to be suitable to providea smooth transition from the segment 306 a to the segment 306 c and togradually transition a migration of analytes from the segment 306 a tothe segment 306 c. For example, in some embodiments, the segment 306 bcan have a length of approximately 5 to 10 mm.

In some embodiments, properties like the inner diameter, length, outerdiameter, and/or thickness of the segments 306 a, 306 b, and/or 306 ccan be selected based on properties of each other and/or on the targetseparation and fractionation of analytes in a sample.

In some embodiments, some important considerations can include thefollowing parameters. (1) Ratio of the inner diameter between the largerinner diameter segment and the smaller inner diameter segment. (2) Ratioof the length of the larger inner diameter segment and the smaller innerdiameter section. In some embodiments the inner diameter of the segment306 a can be based on the inner diameter of the segment 306 c. In someembodiments, the inner diameter of the segment 306 a can be any suitablevalue approximately 2×-3× the inner diameter of the correspondingsegment 306 c. For example, in some embodiments the segment 306 c canhave an inner diameter of 150 μm and the segment 306 a can have an innerdiameter of 450 μm. In some embodiments, the length of the segment 306 a(having a larger inner diameter) can be approximately 3-4 times thelength of the segment 306 c having the smaller inner diameter. In someembodiments, the ratio of inner diameters of segments 306 a and 306 ccan be any number between 2 to 3. In some embodiments, the ratio oflengths of the segments 306 a and 306 c can be the reciprocal of theratio of inner diameters of the segments 306 a and 306 c.

In some embodiments, two or more of the segments 306 a, 306 b, and/or306 c can be formed together as a single capillary tube with sectionshaving variable inner diameter, length, and/or thickness. In someembodiments, the capillary 306 can be formed by joining, coupling, and/or attaching the various segments 306 a, 306 b, and/or 306 c to generatethe capillary 306. In some such embodiments, the coupling can beachieved using any suitable mechanism such that the coupling portion issealed to flow of fluids and electric current, without any juncturebeing used (e.g., a membrane connection which may permit a flux ofcurrent to a separation of analytes within the capillary 306).

In some implementations, the capillary 306 can be configured such thatthe separation of analytes using isoelectric focusing of the chargevariants takes place predominantly within the segment 306 a with thelarger inner diameter. The separated charge variants can be in the formof separated peaks of quantities of charge variants or analytes. Theseparated analytes can then be mobilized to migrate towards the distalend of the capillary 306 using any mobilizing mechanism as describedpreviously. The separated peaks of analytes can be induced to migratefrom the segment 306 a via the transition in segment 306 b and to thesegment 306 c having the smaller inner diameter. The segment 306 b withthe transition portion of the capillary 306 b can be configured to helpreduce or minimize any distortion of the separated peaks during thetransition from the segment 306 a with larger inner diameter and intothe segment 306 c having the smaller inner diameter.

In some implementations the separation using isoelectric focusing can beperformed followed by chemical mobilization. In some implementations,separation using isoelectric focusing can be performed and continuedthrough chemical and/or electrical mobilization such that the analytesare continued to be separated during mobilization.

A separation between two adjacent analytes or charge variants can bemeasured, for example using a probe system, by determining a distancebetween two adjacent peaks of separated analytes, as describedpreviously. A distance between two neighboring peaks can increase as theseparated analytes migrate from the segment 306 a with a larger innerdiameter into the segment 306 c with the smaller inner diameter. Forexample, a peak width can increase with migration from a large diametercapillary segment to a smaller diameter capillary segment in proportionto a ratio of large inner diameter to the small inner diameter. Thedistribution of quantities of each separated charge variant and the peakof the distribution may widen as the separated analyte passes into thesegment 306 c with the smaller inner diameter. In the implementationswhere isoelectric focusing is continued to be carried out duringmobilization, the distribution and the peak of each separated analyte isexpected to re-sharpen during and after migration into the segment 306 cdue to a continuing effect of isoelectric focusing that occurs duringthe mobilization. The separation of analytes in the segment 306 c can beused to guide migration of each analytes or faction to the distal end ofthe segment 306 c and to elute the fraction by collecting into acollection well. The fractions collected by migrating the separatedanalytes to the distal end of the segment 306 c and eluting thefractions can have as high a resolution as desired by modulating areal-time analysis of peak location/migration (e.g., using a probesystem) and using the real-time analysis to guide movement of collectionwells (e.g., using a sample plate assembly system). Such a highresolution can be achieved even for relatively larger samples when usinga capillary 306 with multiple segments as described herein, due to thesegment 306 a with the larger diameter enabling loading of larger sizesamples, and the segment 306 c having the smaller diameter enablinghighly resolved separation and elution of fractions.

When two separated peaks adjacent to each other mobilize from thesegment 306 a with the larger inner diameter into the segment 306 c withthe smaller inner diameter the peak width, the distance between the twopeaks, and the mobilization speed can increase. Experimental resultsindicated that the degrees of increase among these parameters are notproportional. Due to the non-proportional increase of peak width,distance between adjacent peaks, and/or mobilization speed, under thesame elution time, a better purity of fraction could be obtained using acartridge including a capillary with multiple segments each segmenthaving a different inner diameter as shown in shown in FIGS. 17 and 18 .

An additional benefit of using a capillary with multiple segments havingvaried inner diameter is that it allows the use of a longer elutionsegment (which comprises the smaller inner diameter segment). Thisallows the capillary to extend further from the body of the cartridge,as compared to the single segment capillary where a shortened capillaryis preferred, which may benefit from the addition of a vial riser orvial lifter module in the sample plate as shown in FIGS. 7A and 7B,respectively to allow the capillary to reach the vials.

In some embodiments, full-capillary imaging can be used, so that theseparation and mobilization can be monitored in real-time while thesample remains within the imaging window. Eventually, however, theseparation will move out of the imaging window during mobilization andthe last location and speed of a desired peak may have to be used toestimate the arrival of that peak at the distal tip of the elutionsegment of the capillary. As described herein, the electric fieldincreases in the segment of the capillary that has the smaller innerdiameter. The residence time of the sample (e.g., each peak associatedwith each analyte) inside the lumen of the smaller inner diametersegment of the capillary is reduced and the speed of elution of theanalyte is increased proportional to the increase in the electric field.This increased speed of elution and reduced residence time results inthe benefit that a longer section of smaller ID capillary can be usedwhile maintaining the same time delay between a peak passing through theimaging region last used (e.g., to calculate arrival times of elutedanalytes at the distal tip of the capillary) and the distal tip of thecapillary, as for a shorter, single-segment capillary construction.

Cartridge

FIG. 4 is an image depicting an example cartridge 404 to be used with afractionation system, according to an embodiment. The cartridge 404 canbe substantially similar to the cartridge 104, 204, and/or 304 describedherein. As shown in FIG.4, the cartridge 404 includes a running bufferreservoir 405 and a capillary 406 configured to be disposed in therunning buffer reservoir 405. The cartridge 404 includes a porousmembrane disposed at a proximal end of the capillary 406 (not shown,hidden behind the running buffer reservoir 405) a connecting capillary416 configured to be coupled to the capillary 406 via the porousmembrane, and a metal tip 412 disposed at a distal end of the capillary406. The connecting capillary 416 is coupled to a length of deformablepolymer tubing 411 (connection point not visible) which is furthercoupled to an external vacuum source (not shown in FIG. 4 ). Thecartridge 404 also includes a viewing window 414 configured to allowaccess to a portion of the capillary 406 such that contents (e.g.,separated analytes) along the length of the capillary 406 can be probedto detect and analyze a presence and/or location of a desired analyte orfraction.

As shown in FIG.4, the capillary 406 may have to extend sufficientlyfrom the end or bottom edge of the cartridge 404 (e.g., extend for alength greater than 25 mm), such that it may be possible to dispose thedistal end of the capillary 406 into a vial operated by a sample plateassembly of a fractionation system, to extract fractions directly to astandard microtiter plate.

In some embodiments, it may be desirable to minimize the length of thecapillary extending beyond the viewing window to ensure accurate timingduring sample elution and to maximize collection accuracy/efficiency. Anexample of such a cartridge 504 is illustrated in FIG.5.

FIG. 5 is a schematic illustration of a cartridge 504 that can be usedwith a fractionation system, according to an embodiment. The cartridge504 can be substantially similar to the cartridge 104, 204, 304, and/or404 in structure and/or function. The cartridge 504 includes a capillary506. The capillary 506 can be substantially similar in structure and/orfunction to the capillaries 104, 204, 304, and/or 404 described herein.The cartridge 504 includes a porous membrane 513 that can be coupled toa proximal end of the capillary 506, a tubing or connecting capillary511 that can be coupled to the capillary 506 via the porous membrane513, and a metal tip 512 disposed at the distal end of the capillary506. The porous membrane 513, tubing or connecting capillary 511, andthe metal tip 512 can be substantially similar to the respectivecomponents described in detail with reference to cartridges 104, 204,304, and/or 404. The capillary 506 can be situated in the cartridge 504similar to the capillaries 106, 206, 306, and/or 406 with reference tocartridges 104, 204, 304, and/or 404, respectively. The distal end ofthe capillary 506 can be configured to extend past an edge of thecartridge 504 such that the distal end and at least a portion of themetal tip 512 can be dipped or disposed into a well or vial that cancontain a running buffer or a chemical mobilizer as described herein.The cartridge 504 includes a viewing window 514 that permits access fora probe system (e.g., probe system 102 of system 100) to probe and/ordetect a presence and/or location of analytes or desired contents in thelumen of the capillary 506. As described previously, the viewing windowcan be of any suitable size or shape and can be positioned at anydesired location or orientation such that a desired portion or portionsof the capillary can be accessed to be probed.

Example Fractionation System

FIG. 6 is a schematic representation of an example fractionation system600 to separate and elute desired fractions of a sample, according to anembodiment. The system 600 can be substantially similar in structureand/or function to the system 100. The system 600 includes a cartridgeretainer 603 that is configured to retain and use a capillary cartridge604. The cartridge 604 can be substantially similar in structure and/orfunction to any of the cartridges 104, 204, 304, 404, and/or 504described herein. The system 600 can be configured to be used with anyof the cartridges 104, 204, 304, 404, and/or 504. The system 600includes a sample plate assembly 607 that can be substantially similarto the sample plate assembly 107.

The sample plate assembly 607 can be operated to bring one or moresample wells/vials or collection wells/vials such that he distal end ofa capillary in the cartridge can be disposed suitably in thewells/vials. The sample plate assembly 607 can be operated using anysuitable mechanism. In some embodiments, the sample plate assembly canbe operated to move wells or vials can be moved in a lateral,longitudinal, and/or vertical direction. For example, in someembodiments, vials or well can be operated such that individual wells orvials can be advanced in a forward, and backward motion, positioned witha sideward motion, and raised and/or lowered in a vertical motion.

Sample Plate Assembly Configurations

The ability to raise individual vials up into a recess defined in acartridge to allow access to a distal end of the capillary enables thecapillary extension length to be just longer than the depth of fluidthat must be addressed while avoiding interference with adjacent wellsas would be case if using a microwell plate with such a short capillary.FIGS. 7A and 7B illustrate two example sample plate assemblies 707 a and707 b, according to two embodiments. While the process of disposingcapillary in a vial or well is described here by a method of moving thevial or well, in some embodiments, this can be achieved, just as well,by moving the capillary. For example, the cartridge and the capillarycan be mounted on a movable arm that can be actuated to raise and lowerthe distal end of the capillary to a desired extent into a vial or well.

In the embodiments represented by the sample plate assembly 707 a, asdepicted in FIG. 7A, the system can include an actuated vial liftermodule that allows collecting samples into more than just a few widelyspaced, non-interfering, fixed height vials, and keeps the systemcompact. In some implementations, it may be desirable that a user beable to quickly reconfigure an instrument for fraction collection fromanother mode of operation. This can be accomplished with the drop-invial lifter module shown in FIG. 7A that is configured to automaticallyraise vials up to the capillary during the fractionation process. Thevial lifter module can be actuated using any suitable mechanism (e.g.,rotary switch mechanism). In some embodiments, for example vial holderscan be raised and lowered by rotary actuated cams. One or more of thevial locations may contain other reagents (e.g., running buffers,chemical mobilizers, standards, cleaning buffers, etc.) necessary forfraction collection or cartridge cleanup and either positive or negativepressure may be applied to cause fluid flow. In some embodiments. thesample plate assembly (or module) can be removable and insertable into afractionation system as and when needed. Power, communication, andtemperature control can be coordinated from the fractionation system(e.g., via an electronic system) when the sample plate system has beeninserted into a fractionation system. Upon completion of thefractionation process, the user can remove the sample plate assembly torestore previous functionality.

Alternately, as shown in FIG. 7B, in some embodiments, a fixed height,vial holder, with capacity limited due to interferences, could be usedto collect a few samples. The cartridge holder assembly (FIGS. 7A and7B) can be designed such that it does not interfere with any raisedvials yet will provide the functionality required for other modes ofinstrument operation. For example, a presser foot, pipet and a manuallatch release can fully retract or be removed during fractioncollection.

In some embodiments, the cartridge retainer of a fractionation systemmay be reconfigured such that a cartridge retainer does not interferewith any raised vials yet will provide the functionality required forother modes of instrument operation. For example, in some cases, apresser foot, pipet and/or manual latch release can be fully retractedor be removed during fraction collection operation.

FIG. 8 is a flowchart showing an example method 800 of separation andelution of fractions from a sample, according to an implementation. Themethod 800 can be implemented by any fractionation system describedabove including systems 100, 600, and/or by using any capillarycartridge described above including cartridges 104, 204, 304, 404, 504,and/or 604.

At 871, the method 800 includes introducing, at a first time, a samplecontaining a plurality of analytes in a conductive medium into acapillary, ionically coupling a first end of the capillary to a firstrunning buffer having a first pH. The first end of the capillary can bea proximal end of the capillary. In some instances, the running buffercan be an acid held in a running buffer reservoir, as described herein.In some instances, the proximal end of the capillary can be disposed inthe running buffer via porous membrane and coupled to a pressure sourceas described herein.

At 872 the method 800 includes ionically coupling a second end of thecapillary to a second running buffer having a second pH, such that a pHgradient forms along the capillary. In some instances, the secondrunning buffer can be a base held in a sample vial or well operated by asample plate assembly. The second end or distal end can be disposed in awell holding the second running buffer or base.

At 873 the method includes separating, at a second time after the firsttime, at least a subset of the plurality analytes according to theirisoelectric points by applying a voltage across the first running bufferand the second running buffer when the first end of the capillary isionically coupled to the first running buffer and the second end of thecapillary is ionically coupled to the second running buffer. In someinstances, the voltage can be applied via electrical contact points atthe proximal end and the distal end of the capillary as describedherein. In some instances, the contents of the capillary can be analyzedusing any suitable method of detection and/or visualization as describedherein, during and/or after the separation of analytes.

At 874, the method includes placing the second end of the capillary intoa well including a chemical mobilizer at a third time after the firsttime to elute an analyte from the plurality of analytes from thecapillary and into the well. In some instances, the second end or distalend of the capillary can be removed from being disposed in the secondrunning buffer for separation and placed or disposed in a wellcontaining a chemical mobilizer. The chemical mobilizer can have a pHless than or equal to a pI of a fraction of the sample targeted forelution.

At 875, the method includes applying a second voltage across the firstrunning buffer and the chemical mobilizer at the third time and when thefirst end of the capillary is ionically coupled to the first runningbuffer and the second end of the capillary is disposed in the well, thesecond voltage and the chemical mobilizer collectively causing theanalyte to be eluted. The applied second voltage can be configured toelute fractions up to the desired pI. The elution of fractions using asecond applied voltage can implement an electrical mobilization. Thesecond voltage can be the same as, or different from the first voltage.

At 876, the method 800 optionally includes increasing, at a fourth timeafter the third time, a pressure at the first end of the capillary suchthat the pressure induces hydrodynamic flow in the capillary, elutingthe analyte. The elution of fractions using an applied pressuredifferential (e.g., positive pressure) to migrate fractions canimplement a hydrodynamic mobilization.

In some implementations, elution can be achieved using pressure-basedmobilization, chemical mobilization, or electrical mobilization, orhydrodynamic mobilization, or any combination thereof. Optionally themethod can include monitoring during mobilization such that, forexample, the process of mobilization can be ceased or slowed down aftera target peak of a desired analyte or fraction has been eluted.

FIGS. 9A, 9B, and 9C provides example plots showing results fromseparation and isoelectric focusing of a sample containingHER2-Trastuzumab. The plots indicate results obtained using a standardseparation system (FIG. 9A) that may use a standard cartridge, using afractionation system with a cartridge having a single segment capillary(FIG. 9B), and using a fractionation system with a cartridge having amulti segment capillary (with two or more segments having varied innerdiameters). The capillary used for FIG. 9B had a single inner diameterthat was approximately 320 μm. The capillary used for FIG. 9C had atleast one segment (e.g., proximal segment sued for separation) withinner diameter 300 μm and one segment (e.g., distal segment used forelution) with inner diameter of 150 μm. The quality of focusing in FIGS.9B and 9C are shown to be comparable with that by a standard separationsystem in FIG. 9A.

FIG. 10 is a plot showing chemical mobilization of HER2-Trastuzumabusing a fractionation system, according to an embodiment. In the exampleimplementation shown in FIG. 10 , a cartridge with a capillary having asingle segment with inner diameter of approximately 320 μm was used.Each trace from the set of seven traces corresponds to detection andanalysis of the analyte, HER2-Trastuzumab, performed, starting at 0minutes (bottom) and at increasing 5 min intervals for 30 minutes (top).The numbers above each trace represents the mobilization speeds measuredin terms of detector pixels traversed by the pI marker associated withHER2-Trastuzumab at each 5 min interval. The mobilization speedincreases gradually during the initial mobilization phase and stabilizesafter around 15 min under the specific test conditions described above.

FIG. 11 is a plot showing chemical mobilization of HER2-Trastuzumabusing a fractionation system, according to an embodiment. In the exampleimplementation shown in FIG. 11 , a cartridge with a capillary havingmultiple segments was used. The capillary included a segment with alarge inner diameter portion or segment with inner diameter of 300 μmand small inner diameter portion or segment with inner diameter of 150μm. The numbers in the figure are mobilization speeds measured in termsof detector pixels traversed by the pI marker every 5 min, starting at 0minutes (bottom) and ending at 32 minutes (top). The mobilization speedincreases gradually during the initial mobilization phase and stabilizesafter around 15 min under the specific test conditions described aboveand in FIG. 11 .

FIG. 12 provides a plot of traces showing an example of focusing andhydrodynamic mobilization of HER2-trastuzumab using a cartridge with asingle segment long length capillary, according to an embodiment. Thearrow points in the direction of mobilization. The peak resolution iswell-maintained during the hydrodynamic mobilization process as theseparated set of peaks migrates towards the distal end of the capillary.

FIG. 13 provides a plot of traces showing an example of separatedfractions of HER2-Trastuzumab collected from a cartridge with a singlesegment with a large inner diameter (320 μm), according to anembodiment. The unfractionated HER2-Trastuzmab was used as a referencesample (top trace in FIG. 13 ) to identify and match peaks. Thereference sample and the collected fractions were evaluated using theMaurice icIEF method to confirm the pI for each individual fraction. TheHER2-Trastuzmab has 4 main charge variants with approximate pIs 8.5,8.4, 8.3, and 8.2, respectively. The majority of the pI 8.5 chargevariant was collected into well 18 (second trace from the top), themajority of the pI 8.4 charge variant was collected into well 19 (thirdtrace from the top), the majority of the pI 8.3 charge variant wascollected into well 20 and well 21(fourth and fifth traces from thetop), and the majority of the pI 8.2 charge variant was collected intowell 22 (most bottom trace). In an alternative experiment, the majorityof the pI 8.3 charge variant may have been just as well collected in asingle well by adjusting the collection time and/or the speed migrationand elution, as described herein.

FIG. 14 provides a plot of traces showing an example of fractions ofHER2-Trastuzumab collected from a cartridge having a long capillary witha single segment. The unfractionated HER2-Trastuzmab was used as areference sample (top trace in FIG. 14 ). The reference sample and thecollected fractions were evaluated using the Maurice icIEF method toconfirm the pI for each individual fraction. The HER2-Trastuzmab has 4main charge variants represented by peaks 1-4 in FIG. 14 . The majorityof peaks 4, 3, 2, and 1 were collected into wells 5, 6, 7, and 8,respectively, as shown by traces two to six from the top in FIG. 14 . Asaforementioned, by adjusting the time window of fraction collectionbased on the observed peak mobilization speed, a fractionation systemcan be used to selectively collect a single peak into a single fractionor multiple fractions.

Experimental tests were conducted to study icIEF fractionation followedby ZipChip-based mass spectrometry characterization of NIST mAb andXMT-1535 mAb. Individual charge variants of each antibody weresuccessfully collected in less than 2 hours with purity >80% using icIEFseparation conditions with or without urea. Rapid analysis using ZipChipshowed the mass spec identification of major and minor isoformscorrelated well with reported mass spec data (literature and report).Urea in icIEF separation did not affect the quality of fractionation northe mass spec result. Multiple fractionation runs of the NIST mAbsuggested good reproducibility of the system.

FIG. 15 provides a plot of traces showing an example of fractions of aNIST mAb collected from using cartridge with a long capillary having asingle segment. The unfractionated NIST mAb was used as reference samplethe results of which is shown in the top-most trace. The referencesample and the collected fractions were evaluated using the MauriceicIEF method to confirm the pI for each individual fraction. The NISTmAb shows 2 basic charge variants (labeled as b1 and b2 in FIG. 15 ), amain charge variant (labeled as M, tallest peak), and an acidic chargevariant (labeled as a). Well 6 collected the majority of b1 (secondtrace from the top), well 7 collected a significant portion of M (thirdtrace from the top), well 8 collected the remaining portion of M as wellas a small portion of a (fourth trace from the top), and well 9collected the remaining portion of a (bottom most trace).

FIG. 16 provides plot of traces showing an example of the mass spectraobtained from fractions of the NIST mAb collected from using a cartridgewith a long capillary having a single segment. Fractions 6, 7 and 8,indicated in traces labeled S-6, S-7, and S-8, were successfullycharacterized by mass spectrometry. The results from mass spectrometryof each of the fractions 6, 7, and 8 is shown in the insert with eachmass spectra associated with each fraction being indicated by arrows.

FIGS. 17 and 18 show experimental results indicating the benefits ofusing a cartridge with a capillary having multiple segments with variedinner diameter, FIG.17 is plot of traces showing an example of separatedfractions of CD20-Rituximab collected from using a cartridge having acapillary with a single segment with a single inner diameter (320 um).The elution time for fraction collection was 50s. The top most tracerepresents the unfractionated CD20 which was used as reference toidentify and match peaks. The CD20 trace shows two major chargevariants, namely A1 and M, respectively. As shown in FIG. 17 , chargevariant M was collected in fraction F11 (middle trace) and chargevariant A1 was collected over two adjacent fractions F11(middle trace)and fraction F12 (bottom traces). As shown, fraction F11 contains amixture of A1 and M and fraction F12 contains pure A1.

FIG. 18 is a plot of traces showing an example of separated fractions ofCD20-Rituximab collected from using a cartridge having a capillary withmultiple segments. The capillary used included two segments with innerdiameters of 300 μm and 150 μm connected by a transition segment. Theelution time for fraction collection associated with the traces in FIG.18 was 50 s. The top-most trace in FIG. 18 represents the unfractionatedCD20 which was used as reference to identify and match peaks. As in thetop-most trace in FIG. 17 , the CD20 trace shows two major chargevariants, A1 and M. In contrast to the traces representing the fractionsshown in FIG. 17 , however, when using the multi-segment capillary,charge variant M was collected over three adjacent fractions, F14-F16,shown in the second, third, and fourth trace from the top, respectively.Charge variant A1 was collected over two adjacent fractions F17 and F18,shown in the fifth and sixth traces from the top in FIG. 18 . Eachfraction collected using the cartridge with capillary with multiplesegments contained only pure M or only pure A1. Thus, collection offractions with greater purity of analytes or charge variants wasachieved using the cartridge with capillary with multiple segments.

Thus, embodiments disclosed herein present methods and systems toseparate and collect charge variants from a sample using an icIEFprocess. The concentration of a collected fraction can be adjusted byadjusting a starting concentration of a sample, the inner diameter(s) ofthe capillary used, and/or the volume of the chemical mobilizer in thefraction collection wells. Chemicals with different negative ions, forexample, acetate and phosphate, can be used as chemical mobilizers. Thespeed of mobilization can be adjusted by the concentrations of themobilizer, the applied voltage, the composition of the separation buffer(mixture of sample, ampholyte, and additives), and the ID of thecapillary tube. The collection time for a well can be adjusted alongwith the speed of mobilization to allow a single charge variant ormultiple charge variants to be collected into a single well.

Formulations of the assay buffers (e.g., “master mix”) (that may be usedwith other standard separation systems) can be used with the cartridgesdisclosed herein to be used for fractionation. Utilizing similar mastermixes can minimize assay development time needed to transfer an existingassay from the standard icIEF Maurice cartridge to the samplefractionation cartridges and methods described herein (or vice versa).

The methods, apparatus, and systems disclosed herein enablehigh-performance sample fractionation in the easy-to-use Maurice icIEFcartridge format. In contrast to hyphenated methods such as cIEF-MS, avariety of downstream analyses can be performed on the fractionsobtained from the fractionation methods disclosed here. For example, thecollected fraction can be used directly without further samplepreparation; or the collected fraction can be concentrated, diluted, orbuffer exchanged before the downstream analysis; or the same fractionfrom multiple fractionation runs can be pooled together if a largerquantity is needed for certain downstream analysis; or the collectedfraction can be cleaned-up to remove any components incompatible withcertain downstream analysis.

Some embodiments described herein relate to a computer storage productwith a non-transitory computer-readable medium (also can be referred toas a non-transitory processor-readable medium) having instructions orcomputer code thereon for performing various computer-implementedoperations. The computer-readable medium (or processor-readable medium)is non-transitory in the sense that it does not include transitorypropagating signals per se (e.g., a propagating electromagnetic wavecarrying information on a transmission medium such as space or a cable).The media and computer code (also can be referred to as code) may bethose designed and constructed for the specific purpose or purposes.Examples of non-transitory computer-readable media include, but are notlimited to, magnetic storage media such as hard disks, floppy disks, andmagnetic tape; optical storage media such as Compact Disc/Digital VideoDiscs (CD/DVDs), Compact Disc-Read Only Memories (CD-ROMs), andholographic devices; magneto-optical storage media such as opticaldisks; carrier wave signal processing modules; and hardware devices thatare specially configured to store and execute program code, such asApplication-Specific Integrated Circuits (ASICs), Programmable LogicDevices (PLDs), Read-Only Memory (ROM) and Random-Access Memory (RAM)devices. Other embodiments described herein relate to a computer programproduct, which can include, for example, the instructions and/orcomputer code discussed herein.

Some embodiments and/or methods described herein can be performed bysoftware (executed on hardware), hardware, or a combination thereof.Hardware modules may include, for example, a general-purpose processor,a field programmable gate array (FPGA), and/or an application specificintegrated circuit (ASIC). Software modules (executed on hardware) canbe expressed in a variety of software languages (e.g., computer code),including C, C++, Java™, Ruby, Visual Basic™, and/or otherobject-oriented, procedural, or other programming language anddevelopment tools. Examples of computer code include, but are notlimited to, micro-code or micro-instructions, machine instructions, suchas produced by a compiler, code used to produce a web service, and filescontaining higher-level instructions that are executed by a computerusing an interpreter. For example, embodiments may be implemented usingimperative programming languages (e.g., C, FORTRAN, etc.), functionalprogramming languages (Haskell, Erlang, etc.), logical programminglanguages (e.g., Prolog), object- oriented programming languages (e.g.,Java, C++, etc.) or other suitable programming languages and/ordevelopment tools. Additional examples of computer code include, but arenot limited to, control signals, encrypted code, and compressed code.

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Where schematics and/or embodiments described above indicatecertain components arranged in certain orientations or positions, thearrangement of components may be modified. While the embodiments havebeen particularly shown and described, it will be understood thatvarious changes in form and details may be made. Although variousembodiments have been described as having particular features and/orcombinations of components, other embodiments are possible having acombination of any features and/or components from any of embodiments asdiscussed above.

Where methods and/or events described above indicate certain eventsand/or procedures occurring in certain order, the ordering of certainevents and/or procedures may be modified. Additionally, certain eventsand/or procedures may be performed concurrently in a parallel processwhen possible, as well as performed sequentially as described above.

What is claimed:
 1. An apparatus, comprising: a capillary configured toelectrophoretically focus an analyte contained within a sample; arunning buffer reservoir configured to contain a first running bufferhaving a first pH in ionic communication with a first end of thecapillary; a sample plate defining a plurality of wells, the sampleplate and the capillary collectively configured such that a second endof the capillary can move between each well from the plurality of wells;a first well from the plurality of wells configured to contain thesample, the capillary and the sample plate collectively configured suchthat, when a second end of the capillary is disposed in the first well,a portion of the sample can be drawn into the capillary; a second wellfrom the plurality of wells configured to contain a second runningbuffer having a second pH different from the first pH such that when thesecond end of the capillary is disposed in the second well, the bufferreservoir containing the first running buffer and the second wellcontaining the second running buffer are in ionic communication causinga pH gradient to be established across the capillary; an electricalpower source configured to apply a first voltage across the runningbuffer reservoir and the second running buffer such that, when thesecond end of the capillary is disposed in the second well and the firstvoltage is applied, the analyte migrates to a portion of the capillaryassociated with its isoelectric point; and a third well from theplurality of wells configured to contain a chemical mobilizer, and theelectrical power source configured to apply a second voltage, such thatwhen the second end of the capillary is disposed in the third well andthe second voltage is applied , the analyte migrates into the third wellfrom the portion of the capillary associated with its isoelectric pointwhen the capillary is disposed in the second well.
 2. The apparatus ofclaim 1, further comprising: a metal tip disposed over the second end ofthe capillary and configured to be disposed in at least one of the firstwell, the second well, or the third well when the second end of thecapillary is disposed in at least one of the first well, the secondwell, or the third well, respectively, the metal tip configured toprovide electrical connectivity between the second end of the capillaryand the first end of the capillary.
 3. The apparatus of claim 1, furthercomprising: a first electrode electrically coupled the running bufferreservoir and configured to be electrically coupled to the first runningbuffer; and a second electrode electrically coupled to the metal tipsuch that the first electrode, the second electrode, and a portion ofthe sample drawn into the capillary define a portion of an electricalcircuit.
 4. The apparatus of claim 1, further comprising: a porousmembrane disposed on the first end of the capillary, the porous membranebeing configured to allow ion exchange between the first running bufferand the second running buffer when the capillary is disposed in thesecond well, the porous membrane further configured to inhibithydrodynamic flow between the running buffer reservoir and thecapillary.
 5. The apparatus of claim 1, wherein the capillary has aninner diameter of 320-530 μm.
 6. The apparatus of claim 1, wherein thecapillary has an inner diameter of 200-500 μm.
 7. The apparatus of claim1, wherein the capillary has a length of 60-120 mm.
 8. The apparatus ofclaim 1, wherein the capillary has a length of 20-30 cm.
 9. Theapparatus of claim 1, further comprising: a porous membrane disposed onthe first end of the capillary, the porous membrane being configured toallow ion exchange between the first running buffer and the secondrunning buffer and having a molecular weight cut-off from 10 kDa to 500kDa.
 10. The apparatus of claim 1, further comprising: a porous membranetubing disposed on the first end of the capillary, the porous membranetubing being configured to balance ion-exchange between the firstrunning buffer and at least one of the second running buffer or thechemical mobilizer and having a molecular weight cut-off from 10 kDa to500 kDa.
 11. The apparatus of claim 1, further comprising: a porousmembrane tubing disposed on the first end of the capillary, the porousmembrane being configured to balance ion-exchange between the firstrunning buffer and at least one of the second running buffer or thechemical mobilizer, a diameter of the porous membrane tubing beingmatched with an inner and/or an outer diameter of the capillary.
 12. Theapparatus of claim 9, wherein there is no porous membrane tubingdisposed on the second end of the capillary.
 13. The apparatus of claim1, wherein there is no porous membrane tubing disposed on the second endof the capillary.
 14. The apparatus of claim 1, wherein: the voltage isa first voltage; and the electrical power source is further configuredto apply a second voltage across the running buffer reservoir and thechemical mobilizer when the second end of the capillary is disposed inthe third well such that the analyte migrates into the third well fromthe portion of the capillary associated with its isoelectric point. 15.The apparatus of claim 1, wherein: the voltage is a first voltage; theanalyte is included in a first portion of the sample and is focused at afirst distance from the second end of the capillary; the sample includesa plurality of portions including the first portion and a second portionfocused at a second distance from the second end of the capillary, thesecond distance being greater than the first distance such that when thefirst voltage is applied, the first portion migrates to the firstdistance and the second portion migrates to the second distance; and theelectrical power source is further configured to apply a second voltageacross the running buffer reservoir and the chemical mobilizer when thesecond end of the capillary is disposed in the third well such that thefirst portion migrates into the third well from the first distance, amagnitude of the second voltage being determined based on a measure ofseparation between the first distance and the second distance.
 16. Theapparatus of claim 15, wherein the magnitude of the second voltage andthe chemical mobilizer collectively cause the first portion to migratetowards the third well at a rate 1 mm/min to 2 mm/min when the secondvoltage is applied.
 17. An apparatus, comprising: a cartridge; acapillary lumen at least partially disposed within the cartridge anddefined by at least one capillary tube, the capillary lumen configuredto contain an electrically conductive sample that includes a pluralityof analytes, a first portion of the capillary lumen having a first innerdiameter and a first length, and a second portion of the capillary lumenhaving a second inner diameter and a second length; a running bufferreservoir disposed within the cartridge and configured to contain afirst running buffer having a first pH, the capillary coupled to therunning buffer reservoir such that a first end of the capillary lumen isconfigured to be ionically coupled to the first running bufferreservoir; a sample plate defining a plurality of wells, the sampleplate and the cartridge collectively configured such that a second endof the capillary lumen can be moved between each well from the pluralityof wells; a first well from the plurality of wells configured to containa second running buffer having a second pH different from the first pHsuch that when the second end of the capillary lumen is disposed in thesecond running buffer, a pH gradient is established along the capillarylumen; an electrical power source configured to apply a voltage acrossthe running buffer reservoir and the second running buffer such that theplurality of analytes is focused according to their respectiveisoelectric points, generating a separated plurality of analytes; asecond well from the plurality of wells configured to contain a chemicalmobilizer such that when the second end of the capillary lumen isdisposed in the second well, at least one analyte from the separatedplurality of analytes is mobilized into and collected in the secondwell.
 18. The apparatus of claim 17, wherein the first inner diameter is2-3 times that of the second inner diameter.
 19. The apparatus of claim17, wherein the first inner diameter is between 300 and 500 μm.
 20. Theapparatus of claim 17, wherein the second inner diameter is between 100and 200 μm.
 21. The apparatus of claim 17, wherein the first length is3-4 times that of the second length.
 22. The apparatus of claim 17,wherein the capillary tube is monolithically formed.
 23. The apparatusof claim 17, wherein the capillary tube includes a first capillary tubeassociated with the first portion of the capillary lumen joined to asecond capillary tube associated with a second portion of the capillarylumen.
 24. An apparatus, comprising: a cartridge; a capillary includinga lumen at least partially disposed within the cartridge and defined byat least one capillary tube, the capillary lumen configured to containan electrically conductive sample that includes a plurality of analytes;a running buffer reservoir disposed within the cartridge and configuredto contain a first running buffer having a first pH, the capillarycoupled to the running buffer reservoir such that a first end of thecapillary lumen is configured to be ionically coupled to the firstrunning buffer reservoir; and the capillary including a porous membranetubing that couples the capillary, at a first end, to the running bufferreservoir, the capillary including a second end configured to bedisposed in a sample well.